Chemical Science and Technology Laboratory 

Annual Report – FY2003 

NISTIR 7084 
NISTIR 7084 

Chemical Science and Technology Laboratory 

Annual Report - FY2003 

William F. Koch, Acting Director 

Chemical Science and Technology Laboratory

February 2004 

U. S. DEPARTMENT OF COMMERCE 

Donald L. Evans, Secretary

TECHNOLOGY ADMINISTRATION 

Philip J. Bond, Under Secretary for Technology

NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY 

Arden L. Bement, Jr., Director
Compiled and Edited by William F. Koch, Eric B. Steel, Ellyn S. Beary, Joseph M. Conny, and 

Carolyn C. Schwennsen. Cover design by Ann L. Leith. 

For information contact NIST/CSTL 

Telephone: 301-975-8300 

Facsimile: 301-975-3845 

E-Mail: cstl@nist.gov 

Web Sites: www.nist.gov 

www.cstl.nist.gov 

Any mention of commercial products is for information only. It does not imply recommendation or 

endorsement by the National Institute of Standards and Technology, nor does it imply that the 

materials or equipment mentioned are necessarily the best available for this purpose. 

February 2004 
i

Table of Contents 

 Page 

Message from the CSTL Director iii 

A. CSTL Overview 1 

B. CSTL Programs 15 

 1. Automotive and Aerospace (Transportation) 17 

 2. Biomaterials 27 

 3. Pharmaceuticals and Biomanufacturing 31 

 4. Chemical and Allied Products 47 

 5. Energy and Environmental Technologies 61 

 6. Food and Nutritional Products 81 

 7. Forensics and Homeland Security 87 

 8. Health and Medical Technologies 103 

 9. Industrial and Analytical Instruments and Services 119 

 10. Microelectronics 139 

 11. Measurement Standards 147 

 12. Data and Informatics 163 

 13. Technologies for Future Measurements and Standards 171 

C. Exploratory Research 187 

D. Staff Recognition 199 
ii
Chemical Science and Technology Laboratory 

iii

Welcome to the Annual Report of the Chemical Science and Technology 

Laboratory for fiscal year 2003. CSTL is one of the seven disciplinebased laboratories at the National Institute of Standards and Technology.

My name is Bill Koch and I am the Acting Director for CSTL. I have been 

serving in this capacity since July 2003, when Dr. Hratch Semerjian was 

appointed the Deputy Director for NIST. CSTL has been fortunate to have 

had Dr. Semerjian as its Director for over 10 years, and I am grateful to 

him for his insights and visionary leadership that left CSTL a well-trimmed 

ship on a true course; focused, strong and with boundless opportunities. It

has been my privilege to take over the helm, and to move forward with the creative energy that

has always characterized CSTL. 

This past year has been one of change for CSTL. In addition to the changes in leadership that I

just mentioned, there has been the resultant cascade of changes throughout our operating unit. I 

am most pleased at the ease and facility with which CSTL achieved these major changes, a

testament to the depth of its leadership and its dynamic succession planning. Over the course of 

these leadership changes, the concept of teaming to take full advantage of individual strengths

has developed significantly. I am proud of CSTL’s Division Chiefs and our group leaders for their 

dedication, resolve, and creativity. But most of all it has been my honor to lead an outstanding

research staff. It is due to their efforts that CSTL is recognized as the world’s leading metrology 

laboratory in chemical measurements. Today, CSTL has the most comprehensive array of

chemical, physical, and engineering measurement capabilities of any place in the world. Our

activities, products, and services in the national and international metrology community have 

favorably positioned our Nation in the highly competitive global market place – and CSTL’s entire 

staff is to be applauded. Our staff clearly manifests our six core values – Excellence, Leadership

in Metrology, Responsiveness, Objectivity, Respect, and Organizational Commitment. 

We have been challenged in recent years with flat and even decreasing budgets. Therefore, it

has become all the more important to rigorously set our priorities and to focus our efforts on 

projects and programs that will create the largest return for our Nation. We must continue to be

proactive in communicating the breadth and depth of our capabilities as well as the quality and 

impact of our work. Over the past year we have continued to strengthen our interactions with

industry and other stakeholders to assess their needs, and to help us prioritize our work. We are 

grateful for the candid input and ideas these colleagues have shared with us. 

While maintaining our focus on core mission responsibilities of measurement science and 

standards, we are expanding our programs in Nanotechnology, Biosciences and Health, Data

and Informatics, and Homeland Security. It is my pleasure to invite you to look through the main

body of this report, which provides a snapshot of CSTL activities and accomplishments during

fiscal year 2003. The dynamic nature of our programs can hardly be captured in a single 

document, but I hope that what follows will give you a flavor of our work and capabilities, and will

entice you to make full use of our measurement services, and to seek out opportunities for 

collaborations and partnerships with us. 

William F. Koch, Acting Director 

Chemical and Science Technology Laboratory 
Arden L. Bement, Jr., Director 

Hratch Semerjian, Deputy Director

From automated teller machines and atomic clocks to mammograms and

semiconductors, innumerable products and services rely in some way on

technology, measurement, and standards provided by the National Institute 

of Standards and Technology. NIST is a non-regulatory federal agency 

within the U.S. Commerce Department's Technology Administration. 

Founded in 1901, NIST's mission is to develop and promote measurements, 

standards, and technology to enhance productivity, facilitate trade, and 

improve the quality of life. NIST has an operating budget of about $864 million and employs about 

3,000 scientists, engineers, technicians, and support and administrative personnel. About 1,600

guest researchers complement the staff. In addition, NIST partners with 2,000 manufacturing

specialists and staff at affiliated centers around the country. NIST carries out its mission in four 

cooperative programs: 

 

• The Measurement and Standards Laboratories provide technical leadership for vital 

components of the nation's technology infrastructure needed by U.S. industry to continually

improve its products and services. NIST's seven discipline-based Measurement and

Standards Laboratories work at all stages of the pipeline from advancing basic science and

pioneering new measurement methods to the development of standard test methods,

materials, and data to ensure the quality of commercial products. The seven NIST 

Laboratories are: Electronics and Electrical Engineering Laboratory (EEEL), Manufacturing 

Engineering Laboratory (MEL), Chemical Science and Technology Laboratory (CSTL),

Physics Laboratory (PL), Materials Science and Engineering Laboratory (MSEL), Building and 

Fire Research Laboratory (BFRL), and Information Technology Laboratory (ITL). 

• The Advanced Technology Program (ATP) bridges the gap between the research lab

and the market place, stimulating prosperity through innovation. Through partnerships with 

the private sector, ATP’s early stage investment is accelerating the development of innovative 

technologies that promise significant commercial payoffs and widespread benefits for the

nation. 

• The Manufacturing Extension Partnership (MEP) was established in 1989 as the first 

federally funded extension centers to help small manufacturers improve their capabilities and

performance, which is a necessity for survival in the global marketplace. Today, the

Manufacturing Extension Partnership is a nationwide network of more than 400 not-for-profit

centers and field offices. The centers, serving all 50 States and Puerto Rico, are linkied

together through NIST. MEP makes it possible for even the smallest firms to tap into the 

expertise of knowledgeable manufacturing and business specialists all over the U.S. 

• The Baldrige Quality Program is an outreach program associated with the Malcolm

Baldrige National Quality Award that promotes performance excellence among U.S.

manufacturers, service companies, educational institutions, and health care providers;

conducts outreach programs and manages the annual Malcolm Baldrige National Quality 

Award which recognizes performance excellence and quality achievement.

iv

http://www.nist.gov
 Acting Director 

 William F. Koch 

Acting Deputy Director 

 Eric B. Steel 

A. Overview 

The Chemical Science and Technology Laboratory (CSTL) of the National Institute of Standards 

and Technology (NIST) is the United States’ reference laboratory for chemical measurements.

CSTL is entrusted with developing, maintaining, advancing, and enabling the chemical measurement system for the U.S., thereby enhancing U.S. industry’s productivity and competitiveness, 

assuring equity in trade, and improving public health, safety, and environmental quality. With current world events, the need for accurate and timely chemical and biological measurements is 

more important than ever. CSTL is ready to meet these new challenges.

Today as part of the Commerce Department’s Technology Administration, NIST’s vision is to be

the global leader in measurement and enabling technology, and delivering outstanding value to

the nation. NIST provides scientific leadership for the Nation's measurement and standards infrastructure and ensures the availability of essential reference data and measurement capabilities. 

To discharge these responsibilities, NIST maintains expertise in a broad range of science and 

technology areas. The Chemical Science and Technology Laboratory is responsible for measurements, data, and standards in chemical, biochemical, and chemical engineering sciences. 

Building on a hundred-year history of technical excellence, today’s CSTL has the most comprehensive array of chemical, physical, and engineering measurement capabilities and expertise of 

any group worldwide working in chemical science and technology. 

NIST’s Vision 

Preeminent Performance: Future 

economic competitiveness, national 

security, and public well-being will 

be shaped by revolutionary developments in the biosciences, 

nanoscience, and information and 

knowledge management – a transformation enabled by NIST’s unique 

measurements, standards, and enabling technologies and services. 

NIST’s Mission 

... is to develop and promote measurement, standards, and technology

to enhance productivity, facilitate 

trade, and improve the quality of 

life. 

CSTL’s Vision is to be a world-class research laboratory 

that is recognized by the nation as the primary source for

the chemical, biochemical, and chemical engineering

measurements, data, models, and reference standards

that are required to enhance U.S. industrial competitiveness in the world market. 

CSTL’s Mission is to serve as the nation’s Reference 

Laboratory providing the chemical measurement infrastructure to:

• enhance U.S. industry's productivity and competitiveness;

• assure equity in trade; and 

• improve public health, safety, and environmental 

quality. 

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CSTL’s programs have been guided by a formal Strategic Plan for more than a decade. Periodically, CSTL’s Strategic Plan is reassessed and updated to respond to National priorities, changing customer needs, and the rapid evolution of science and technology. This process assures 

continued alignment with the similarly evolving NIST 2010 strategic plan. Although the details of 

the strategic planning process have varied over the years, its essential characteristics — customer-focus and openness to all stakeholders — have been preserved. CSTL’s Strategic

Plan focuses on achievement of three strategic goals: 

Measurement Standards: Establish CSTL as the pinnacle of the national traceability and international comparability structure for measurements in chemistry, chemical engineering, and 

biotechnology, and provide the fundamental basis of the nation’s measurement system. This 

objective is achieved by: 

• Developing and demonstrating international comparability for chemical and physical measurements, 

• Supporting and strengthening the traceability structure in the U.S., and 

• Supporting and strengthening voluntary standards organizations.

Chemical and Process Information: Assure that U.S. industry has access to accurate and reliable data and predictive models to determine the chemical and physical properties of materials

and processes. This objective is realized by: 

• Developing benchmark data for the properties of important substances, classes of substances, and systems; 

• Developing data collections, data prediction methods, and models to meet high-priority industrial and national needs; 

• Contributing to the development of consensus standards for key properties, substances,

and processes; and 

• Developing procedures or protocols for data access, facilitating data exchange, and disseminating formatted data.

Measurement Science: Anticipate and address next-generation measurement needs of the 

Nation. This final goal is achieved by: 

• Maintaining a strong and cutting-edge research program to support the Nation’s measurement and standards infrastructure, and 

• Establishing new measurement capabilities to support new and advanced technology development and dissemination. 

2
Organizational Structure and Resources 

CSTL is organized to reflect the technical expertise that is the foundation of our technical programs and allows us to accomplish our mission. The Laboratory consists of five Divisions: the 

Biotechnology Division, the Process Measurements Division, the Surface and Microanalysis Science Division, the Physical and Chemical Properties Division, and the Analytical Chemistry Division. Each Division employs a group structure organized to achieve synergy and critical mass in 

its technical program areas. 

While the divisional structure has remained stable over the past year, there have been significant

changes in leadership, and these are reflected in the organization chart below. Since July 2003 

Hratch Semerjian, CSTL’s director of 11 years, began serving as Deputy Director of NIST, and

Bill Koch, CSTL’s Deputy Director, was named Acting Director of CSTL. The vacated position is

now held by Eric Steel, a Group Leader from the Surface and Microanalysis Science Division. 

John Small moved into the vacated Group Leader position. Unrelated to this cascaded change in

leadership is the appointment of Fred Schwartz as Group Leader of the Structural Biology Group 

in the Biotechnology Division, a position vacated by Gary Gilliland. The facility with which CSTL

achieved these major changes is a testament to its successful succession planning. In addition

the concept of teaming to take full advantage of individual strengths has developed significantly. 

To achieve its goals, CSTL maintains an experienced, well-educated professional staff. The fulltime permanent staff numbered 265 in FY03, in addition to 85 temporary and part-time employees. Also, there were 237 guest researchers working closely with CSTL staff in various aspects of

the research program. The technical capabilities of CSTL staff are extensive; they hold degrees in

chemistry, physics, engineering, biology, and computer science. A capable technical support staff 

augments the professional staff. Approximately 80 % of the technical staff has PhD degrees. 

3
CSTL Funding, FY 2003

Other Agency

15 %

Competence 

5 %

ATP 3 %

Research Base

66 %

 Measurement Services

Funding Sources 12 %

Total = $67.2M

CSTL Staff, FY 2003

Engineers, 

 Comp. Scientists 

(37) 

Physicists 

(45) 

Technical 

Support (30) 

Administrative

Support (36) Chemists 

(152)

Biologists, 

Biochemists (28)

Students (22)

CSTL’s physical facilities are located at 

the major NIST sites in Gaithersburg, 

Maryland and Boulder, Colorado, as

well as at the Center for Advanced Research in Biotechnology (CARB) in

Rockville, Maryland and the Hollings 

Marine Laboratory (HML) in Charleston, 

South Carolina. The Physical and

Chemical Properties Division has operations located at the NIST sites in

Gaithersburg and Boulder. Biotechnology Division staff work closely with that 

of the University of Maryland Biotechnology Institute located at CARB. The Analytical Chemistry 

Division is the primary liaison with HML, having staff located in Charleston. HML is a cooperative

research facility involving NIST, the National Oceanic and Atmospheric Administration, the South 

Carolina Department of Natural Resources, the University of Charleston, and the Medical University of South Carolina. 

Hollings Marine Laboratory

The Analytical Chemistry and Biotechnology Divisions have now been located in the Advanced 

Chemical Sciences Laboratory (ACSL) for over 

four years, the first new research facility on the

NIST Gaithersburg Campus in over thirty years.

Although ACSL adds significantly to CSTL’s research capabilities, it does not meet all facility

needs of our program. 

NIST is proceeding with implementing its facilities master plan, the current focus of which is the Advanced 

Measurements Laboratory (AML). CSTL is scheduled 

to move into the AML during spring of 2004. This new 

facility will accommodate the Surface and Microanalysis Science Division, as well as the Pressure and 

Temperature activities in the Process Measurements 

Division. Attention is now being directed to improving

the facilities in Gaithersburg and Boulder not located in 

the ACSL and AML. 

4
CSTL Strategic Directions 

In a recent review of our strategic plan, the CSTL leadership defined five major thrusts areas. 

These thrusts define our unique role and channel our core competencies to address existing and 

anticipated stakeholder needs, and cross-cut organizational lines. 

1. Nanometrology: for chemical characterization,

device characterization, and processes at the submicrometer scale 

2. Properties Information Infrastructure: technologies 

for generation, evaluation, and dissemination of

chemical and physical property information

3. Biometrology: for biochemically- or cell-derived

materials, devices, or processes 

4. Process Metrology: for chemical engineering 

processes 

5. Chemical Metrology: for the chemical 

characterization of materials and complex mixtures 

These are, in name and perhaps emphasis, a slight departure from the five thrust areas that

CSTL identified and invested in since 1997: Biotechnology, Nanotechnology, Healthcare, Data

and Informatics, and International Measurement Standards. We have chosen to focus on the 

metrological aspects of these emerging and changing 

areas to more clearly describe to our staff the specific 

role CSTL should play. NIST has also renamed its 

Programmatic Strategic Focus Areas (SFAs) to emphasize the metrological role of NIST. It is fair to say

that Nanometrology Information/Knowledge Management, and Biosystems are infrastructural, while Homeland Security and Heath are more applied areas. 

It is critically important to ensure that CSTL programs and projects are dynamic and flexible 

enough to respond to national and international technology trends, and the changing needs of our 

customers and stakeholders. CSTL has several mechanisms in place that facilitate the development of new projects at the Division, Laboratory and NIST levels. Each year, CSTL Management

reviews its portfolio of programs and projects, and considers proposals for high priority projects. 

Priority is given to new projects that closely align with NIST and CSTL strategic goals. 

NIST Programmatic SFAs 

Homeland Security 

Nanometrology/Nanotechnology 

Biosystems and Health 

Information/Knowledge Management 

CSTL core competencies are the scientific expertise and infrastructure in

measurement science, measurement 

standards, and properties data that

provide the stability and agility to 

meet our customers’ needs. CSTL 

views these competencies as critical 

to the future viability of our organization. They are dynamic and relevant, 

enabling new as well as promoting 

change in existing technologies, and 

leave CSTL and NIST poised to meet

both current and next generation

measurement and standards needs. 

5
Selection of new projects for reprogramming and identification of projects to be phased out require specific criteria. CSTL has used the following criteria for its decision-making: 

• Industrial Need – Magnitude and immediacy of industrial need 

• Match to Mission – CSTL provides the Nation’s chemical measurement infrastructure

• Making a Difference – Is the CSTL contribution critical for success? 

• Nature and Size of Impact – Measure of anticipated impact relative to investment (rate of 

return) 

• Timely Quality Output – The ability to respond in a timely fashion with high-quality output 

• Science/Technology Opportunity – Recent scientific and technological advances present 

new opportunities 

To provide our staff with stimulus for innovative ideas and new areas of research, CSTL continues its tradition of one-year funding for small seed projects that evaluate the feasibility of new 

technologies and measurement methods. In FY2003, CSTL funded 11 such projects, following a

rigorous internal peer review process. Progress reports for these projects are included in Section 

C of this Annual Report. 

CSTL Programs 

CSTL is a multifaceted, synergistic organization with a unique customer base. Perhaps the most

remarkable aspect of the CSTL customer-base is its breadth. The primary customers we serve

are: industry; federal, state, and local government agencies; standards and industrial trade organizations; and the academic and scientific communities. 

U.S. industry is the largest consumer of our products and services. These customers come from

established industrial sectors, such as the chemical manufacturers, and emerging industries,

such as biotechnology and nanotechnology. The products and services they rely on are as varied 

as Standard Reference Materials (SRMs), Standard Reference Data (SRDs), calibration services, 

and novel measurement methods. Reference materials and calibrations provide traceability to the 

International System of Units (SI), which is essential to fair trade, improved reliability of measurements, and regulatory compliance. Data compilations facilitate modeling of chemical processes and rational product design. New measurement techniques ensure improved product

quality and process efficiency, and in turn enhance competitiveness. 

Federal, state, and local governments are another important consumer of our products and services. These agencies use our products and services in ways similar to the commercial sector, 

but in different arenas. For example, calibrations, evaluated data, and reference materials are

used to ensure the reliability of environmental monitoring programs. Carefully characterized

physiological samples lend credibility to forensics tests. Measurement technologies developed 

and perfected in CSTL verify compliance with international treaties. Other national laboratories

exploit the expertise and techniques developed here in discharging their mandates. 

A final group that uses our expertise includes trade organizations, standards committees, and the 

academic and scientific community. These groups rely on CSTL’s expertise for advice and guidance in establishing practical standards and uniform protocols. The academic and scientific

communities rely on the data produced and compiled by our staff as touchstones for their own

research. 

6
CSTL’s programs are customer-based and

serve to focus our activities. The agility of our

technical staff permits CSTL to respond in a

timely manner to changes in customer needs 

within each industry segment, changes in priorities among industries as well as National priorities. CSTL has identified 10 programmatic 

areas aligned with industrial segments and National priorities, and these are highlighted in

Section B of this report. It is clear that those

projects listed contribute to strengthening the

metrological infrastructure of each industry sector. Affirmation and, if needed, modification of this 

structure is made yearly based on needs assessments and stakeholder input. Any modification is 

a reflection of CSTL’s current level of activity with specific industries, and captures changes in

priority both internal and external. 

1. Automotive and Aerospace

2. Biomaterials

3. Pharmaceuticals and Biomanufacturing

4. Chemical and Allied Products

5. Energy and Environmental Technologies 

6. Food and Nutritional Products

7. Forensics and Homeland Security

8. Health and Medical Technologies

9. Industrial and Analytical Instruments and 

Services

10. Microelectronics

It is also beneficial to articulate CSTL projects 

by activities that cross-cut industry sectors.

Therefore, CSTL has three cross-cutting programs that are clearly aligned with CSTL’s 

three strategic goals. 

1. Measurement Standards 

2. Data and Informatics 

3. Technologies for Future Measurements 

and Standards 

Measures of Success 

CSTL employs many mechanisms to capture feedback on the quality, relevance and impact of 

our programs and activities. In its mandated review of NIST programs, the National Research 

Council (NRC) Board on Assessment (BoA) Panel for CSTL provides a rigorous peer review of 

the quality of our technical activities. The Panel is comprised of technical experts from industry,

academia, and national laboratories covering the broad range of disciplines corresponding to

CSTL program areas. Direct customer feedback on the quality of our work, external recognition 

of our technical staff, the leadership roles of our staff in standards activities, and the performance 

of CSTL in Key Comparisons conducted among other countries’ National Metrology Institutes,

under the auspices of CIPM Consultative Committees, provide further direct evidence for the

quality and relevance of our technical programs. Our performance in CIPM Key Comparisons in a

broad range of measurement fields, where our performance is compared to the best laboratories 

of the world, is an excellent measure of our capabilities and an opportunity to demonstrate our

global leadership. 

CSTL also organizes many workshops and meetings to assess the needs of specific industry 

segments, and to prioritize these needs to address the highest impact areas. Workshops, which 

bring together experts from NIST, industry, academia, and other national agencies, are a particularly useful and cost-effective means of developing competent technical strategies for meeting the 

NIST mission. CSTL interactions with other federal agencies also provide us with invaluable 

knowledge on measurements, standards and data needs that may result from promulgation of 

new regulations, or new more sensitive and selective measurement methods that may be needed

to improve the science base needed for decision making. Direct customer and stakeholder feedback on the relevance of our current work and anticipated measurement needs of industry is also 

sought out through ongoing interactions. This information is used to help develop programs in

appropriate directions and assure that CSTL programs are effectively meeting the needs of our 

customers. 

Other direct indicators of the relevance of our work are CSTL outputs, such as new calibration 

services, SRMs, SRDs, publications, invited talks, patents, licenses, the number of companies 

7
using our calibration services, the number of institutions who purchase our Standard Reference 

Materials (SRMs) in the U.S. and abroad, and the number of institutions who purchase or license

our Standard Reference Data (SRD) products, or download our web-based databases. The quality of Measurement Service delivery is another important performance measure for our customers. We constantly monitor and strive to improve the turnaround time of our calibration services.

We have been successful in reducing the certification times of our SRMs (to less than two years),

and improving our prioritization process to reduce out-of-stock items. CSTL is also making special efforts to expand our web-based data dissemination efforts, and to provide more standardized formatting and single-portal access to all CSTL databases. 

Some of the CSTL outputs and interactions in FY2003 are summarized in the following table. 

Selected CSTL Outputs – FY 2003

Div. Pubs. 1 Talks Committees

2 Seminars Conferences CRADAs Patents 

Issued 

SRMs

/RMs3

SRDs Cals.4

830 1 17 28 8 4 0 0 0 0 0 

831 85 98 42 5 21 1 2 3 1 0 

836 33 72 96 15 5 1 4 0 0 1140 

837 43 93 74 6 12 2 0 1 4 0 

838 82 114 100 16 1 0 1 0 17 2 

839 88 178 127 20 11 0 0 67 1 359 

Total 332 572 468 70 64 4 7 71 22 1501 

1 Publications appearing in print in FY2003. Another 223 manuscripts have been submitted for publication. 2

Committee totals include 64 editorships and TRC 3

CRADA’s signed this year only 4

Includes four licensed. There are a total of 38 active patents. 5

SRMs/RMs (Certificates issued) 

6

 Ca librations were performed for over 160 customers 

Division Key: 

830 Laboratory Office 837 Surface and Microanalysis Science Division 

831 Biotechnology Division 838 Physical and Chemical Properties Division 

836 Process Measurements Division 839 Analytical Chemistry Division

SRM – Standard Reference Material SRD – Standard Reference Data 

---------------------------------------------------------------------------------------------------------- 

8
Lastly, economic impact studies, conducted by outside experts, investigate the impact of CSTL

products and services on the U.S. economy, thereby providing a quantitative tool for measuring 

the influence and reach of our programs.

Evaluations of the economic impact of NIST's metrology programs in specific technical areas are 

carried out through NIST-commissioned studies, performed by external contractors. These studies provide both qualitative assessments and quantitative estimates of the economic impacts resulting from the several categories of technology infrastructure that NIST provides to U.S. 

industry. Quantitative estimates are provided either as benefit-to-cost ratios or as rates of return

to the nation (social rate of return). The results of these impact assessments not only respond to 

the need to measure and analyze current and past performance but also contribute to future strategic planning. In FY2003, we completed a study of the impact of measurement uncertainties on

healthcare costs, described in some detail immediately below. In addition, we summarize some of

the results of earlier impact studies, with updated information on subsequent activities and benefits accrued since the publication of each report.

Standards for Calcium Testing: 

NIST and the Mayo Clinic in Rochester MN jointly commissioned a 

study to evaluate the impact of clinical laboratory measurement

variations on both patient care and healthcare costs. The issue of

measurement variability is accentuated in recently developed integrated healthcare networks, which were designed to help reduce clinical practice variation. In this 

study mathematical models were designed and data analyzed from over 89,000 patients receiving calcium tests at the Mayo Clinic between 1998-1999. This data analysis was done in order to

evaluate the impact of laboratory variation on total healthcare costs to help illustrate the importance of traceable standards and robust reference methods. Based on interviews with laboratory 

managers and equipment manufacturers, it was determined that calibration error has the potential

to cause a bias of 0.1 mg/dL to 0.5 mg/dL in up to 15 % of the 

calcium tests. The study revealed that patients with elevated or 

borderline calcium levels resulted in increased heath care

costs of up to $31 per patient for an analytical bias of 0.1

mg/dL and up to $89 per patient when the bias was 0.5 mg/dL.

In the U.S., with approximately 3.55 million patients per year 

receiving serum calcium tests that are affected by systematic 

bias, the potential economic impacts range from $60 million to 

$198.8 million for analytical biases of 0.1 mg/dL and 0.5

mg/dL, respectively. 

This is the first of a threepart study, and the impact of 

measurement uncertainty on 

PSA measurements is underway, to be followed by a 

similar study for uncertainties related to cholesterol

measurements. 

NTRM Gas- Mixture Standards: 

The NIST Traceable Reference Materials (NTRM) program was created to 

help address the problem of increasing needs for reference materials with a

well-defined linkage to national standards. An NTRM is a commercially 

produced reference material with a well-defined traceability to existing NIST

standards for chemical measurements. This traceability linkage is established

via criteria and protocols defined by NIST and tailored to meet the needs of 

the metrological community to be served. The NTRM concept has been 

implemented in the gas standards area to allow NIST to

respond to increasing demands for high quality reference 

materials needed to implement the "Emissions Trading" 

provisions of the Clean Air Act Amendment of 1990. 

The NTRM program was created by NIST in collaboration with the U.S.

9
Environmental Protection Agency (EPA) and the specialty gas companies (SGCs) to increase the 

availability of NIST-certified reference materials. Under the program, SGCs follow NIST technical 

guidance to manufacture SRM-equivalent standards and submit these standards to NIST for

certification. Once certified, the NTRMs are the functional equivalent of SRMs and are used to

assay the large volume of secondary reference standards demanded by consumers to meet 

regulatory requirements. For this impact study, SGCs were

surveyed, as well as end users such as electric utilities,

transportation equipment firms, petrochemical firms, commercial laboratories, and government agencies. Since 

NTRM program inception in 1992, 15 specialty gas 

companies have worked with NIST to certify over 8600

NTRM cylinders of gas mixtures that have been used to 

produce approximately 500,000 NIST-traceable gas standards for end users, with a market value of about $140

million. Over the past decade, this process has been and 

continues to be an integral component of the high 

accuracy reference gas supply chain. According to the economic impact study published in August 2002, the social rate of return of this program is 225 %, with a benefit-to-cost ratio estimated

to be 24 to 1, and a net present value of $56 million. 

Currently NIST is working with 

the SGCs to reduce the uncertainty in the NTRMs. This is 

driven by market demand for a 

“better product”; improvement 

in bottom-line profit by avoiding 

redundant analyses; and desire 

to anticipate future changes in 

regulatory requirements particularly as they relate to emissions 

trading. 

Cholesterol Standards: 

Chemical metrology is at the heart of accurate medical diagnosis 

and the development of measures to improve our health and

ensure long life. In the U.S. more than one trillion dollars are 

spent each year on health care, which is about 14 % of the U.S. 

GDP. More than 20 % of these expenditures are for 

measurements. 

Over the last three decades, NIST, in cooperation with the College of American Pathologists 

(CAP), has developed a series of highly accurate and precise methods for a number of clinically 

important serum constituents, including cholesterol. These methods are recognized by the international clinical laboratory community as "definitive" and have been used to certify a series of 

cholesterol SRMs. The first pure crystalline cholesterol (SRM 911) was introduced in 1967. Using

the definitive method, serum cholesterol SRMs were developed in 1981 (SRM 909) and again in

1988 (SRMs 1951 and 1952). These SRMs have led to a steady decrease in the measurement

uncertainty from 18 % relative in 1969 to about 6 % in 1999. The economic consequences of

NIST’s Cholesterol Standards Program are experienced at several levels of the supply chain from

manufacturers, to network laboratories, to clinical laboratories that ultimately deliver medical services to the consumer. The results of this study indicate that NIST has played an important economic role in support of a national effort to monitor, measure, and control cholesterol levels,

thereby contributing to the reduced level of heart disease. The economic impact study estimates 

a benefit-to-cost ratio of 4.5, and a social rate of return of 154 %. The net present value was calculated to be more than $3.5 million. This report was published in September of 2000, and the

timeline extended from 1986 to 1999. 

Recent Developments: NIST Cholesterol SRMs remain an

important part of the traceability chain for clinical laboratories and manufacturers of cholesterol measurement kits. 

Approximately 500 SRM units were sold per year over the 

past three years. 

Cholesterol SRMs have continued to play a critical role in the 

steady decrease in the measurement uncertainty from 18 %

relative in 1969 to about 6 % in 

1999, and 3 % today.

Beyond Cholesterol: These Cholesterol SRMs, along with NIST’s suite of clinical standards, 

also serve as important “high order” reference materials, providing measurement traceability for in 

10
vitro diagnostic (IVD) products. This level of traceability is required for IVD products to be imported into European markets, as specified in the EU IVD Directive that went into effect in December of 2003. Since U.S.-based companies supply 60 % of the IVD products sold in Europe, it

is critical to U.S. industry that NIST maintain and expand SRMs that provide the required traceability. Specifically, the need for manufacturers to provide traceability statements to their customers and to calculate uncertainty for their device calibrators has its origins in this IVD Directive. In

order to help manufacturers meet the implementation deadline, NIST’s CSTL and ITL collaborated with industry representatives to develop and offer a widely accessible and broadly disseminated web cast workshop to provide the tools needed by the industry to perform uncertainty

calculations for their calibrators and controls. The uncertainty workshop was presented before a

live audience on the NIST Gaithersburg campus and was also simultaneously web cast around

the world and viewed by over 150 participants. It was made available on the web for future viewing in CSTL archives. Since its availability, nearly 1000 clips from the workshop have been replayed.

Sulfur in Fossil Fuels: 

The sulfur content of fossil fuels is one of the most important intrinsic factors that determine fuel 

prices. The accurate determination of the sulfur concentration in fossil fuels is required as a result 

of environmental regulation that places increasingly lower limits on their sulfur content and the 

imposition of large fines for non-compliance. At every stage in the process (mining,

transportation, buying and selling, and combustion) the sulfur content of both oil and coal must be

determined in order to meet buyer and seller specifications that are dictated in large part by 

government environmental regulations. The efficient and cost effective movement of coal and oil 

from the mine and well to power plants and refineries requires precise and accurate 

determination of sulfur content in two or more laboratories. For equity in trade and the efficient

production of energy, it is mandatory that instrumentation in these laboratories be calibrated using 

accurate standards. 

NIST has certified the sulfur content in about 30 coal and fuel oil SRMs to an accuracy of better 

than ± 0.1 % relative, using isotope dilution mass spectrometry. These SRMs provide industry 

with the primary calibration materials needed for instrumentation used in routine measurements.

SRMs also provide industry with a strong traceability link to NIST for such measurements,

whether they be for setting the price of fuel or for demonstrating compliance with environmental

regulations. Surveyed industry representatives indicated that NIST SRMs have decreased the

level of uncertainty associated with their measurements of sulfur content. This reduction has led 

to economic benefits throughout the supply chain. Included in the measures of economic benefits 

are improvements in product quality, production efficiency, and reductions in transaction costs

and sulfur emissions to the environment. This study, published in February 2000 estimates a 

benefit-to-cost ratio of 113, and a social rate of return of 1,056 %. The Net Present Value was 

calculated to be more than $400 million. The time 

period studied was from 1984 to 1999. 

Recent Developments: The petroleum refining

industry faces a June 2006 deadline to comply with 

stringent limitations on the sulfur content of highway diesel fuel. By this date, most refiners must

meet a 15 μg/g or parts per million (ppm) standard 

for at least 80 % of the highway diesel fuel produced, with a 500 μg/g (ppm) cap on the remaining 

20 % of their production. By 2010, all highway diesel fuel must meet a 15 μg/g (ppm) cap. In addition, the industry expects that the EPA will propose 

similar regulations for the non-road diesel fueled

A new twist on existing standards: 

New environmental regulations have

capped Hg emissions at 26 tons starting 

in 2010 and reduced to 15 tons by 2018. 

There has been value-added to NIST’s

eight natural coal SRMs that were originally certified only for S and now have

been reanalyzed and certified for Hg. 

These new certifications will take on significant importance since they will be 

used increasingly by the electric power 

industry to ensure compliance with these 

new regulations.

11
vehicles. The benefits to the industry in the 2000 Economic Impact report were derived from improved production efficiency in producing diesel fuel that complied with the 500 μg/g (ppm) sulfur

cap. The new cap of 15 μg/g (ppm) sulfur puts additional constraints on the industry that translate to higher potential cost avoidance by the use of SRM standards. In December 2003, NIST

released a new low-level diesel fuel SRM with a sulfur concentration of 11 μg/g that will meet the 

new need of the industry. In addition, a kerosene SRM (SRM 1616b) is under development with

sulfur levels at approximately 8 μg/g, the lowest level of any Certified Reference Material available worldwide. This material is being used in a NIST-led pilot study now underway under the 

auspices of the Comité International des Poids et Mesures (CIPM). 

Alternative Refrigerants: 

Occasionally, an accelerated R&D program must be undertaken to respond to industry needs that

are constrained by set deadlines. With the timetable imposed by the Montreal Protocol of 1987 as

an incentive to develop new alternatives to CFCs, NIST engaged in 

research that would allow industry to make the switch to alternative 

refrigerants in a timely and economical fashion. NIST began by identifying the basic requirements for new refrigerants according to the new 

rules, and then started research on determining the physical properties 

of such candidate alternatives. NIST’s most effective form of information dissemination has been the REFPROP program, a computer software package that is available through NIST’s Standard Reference

Data Program. The REFPROP program enables manufacturers and 

users of alternative refrigerants to model the behavior of refrigerant 

mixtures in their respective manufacturing processes, a key method in

developing CFC replacements. A comparison of industry benefits with the funding stream of

NIST’s research program estimated a social rate of return of at least 433 %, and a benefit-to-cost

ratio of 4 to 1. This report was published in January 1998, and the period studied was from 1987 

to 1996. 

Recent Developments: The NIST REFPROP database has been updated several times since

the economic assessment, and version 7.0 was released in August 2002. This database continues to be the primary technology transfer vehicle for the continuing work of CSTL on refrigerants.

The latest version includes additional fluids and mixtures, improved property models, increased 

calculational speed, and an enhanced user interface with additional properties and calculation

and plotting options. Its position as the de facto standard in the refrigeration industry is stronger 

than ever. In addition to direct use by engineers in industry (it is the number two seller among the

databases distributed by the NIST Standard Reference Data Program), it provides the core property routines for the CYCLE-D and REFLEAK databases distributed by NIST as well as packages 

distributed by several refrigerant and equipment manufacturers to their customers. The de facto

status will likely become official in the coming year: a draft ISO standard on refrigerant properties

specifies the same models (and thus the same property values) as REFPROP. Although

REFPROP is not explicitly specified in the standard, the

ISO Working Group recognized the high quality and wide 

use of REFPROP and adopted the same models. A new

title for version 7 of REFPROP (Reference Fluid Thermodynamic and Transport Properties) with “Reference Fluid”

replacing the “Refrigerant” of earlier versions indicates our 

direction to provide a single database would encompass 

refrigerants, cryogenic fluids, hydrocarbons, and simple

inorganics. At this time, Version 7.1 of NIST REFROP is scheduled for release in the summer of

2004, and would incorporate NIST14 capabilities. Plans to incorporate the NIST12 capabilities 

will follow. 

DuPont phased out their own “Refrigerant Expert” package in 1999, 

and pointed their customers to 

REFPROP and CYCLE-D, and 

REFPROP has been licensed for 

use with the ASPEN process 

simulation package. 

12
Thermocouples:

The NIST thermometry program includes both calibration services and research on thermocouples. Thermocouples are among the most commonly used sensors for monitoring and control of

manufacturing processes. The annual sales of thermocouple products sold by the U.S. thermocouple industry (suppliers of wire and thermocouple assemblies) into the U.S. market are approximately $280 million. The incorporation of these devices into higher levels of product 

structures across a broad base of domestic industries affects a much larger portion of the manufacturing sector, estimated to be on the order of 

$80 billion. 

Benefits were estimated based on surveys and 

interviews of the thermocouple industry and did not

include the much larger, though diffuse, community 

of device users. Participants were asked to estimate the additional expenses that would have 

been incurred if NIST were to cease to provide

primary calibration services. NIST expenditures 

included research on the fundamental and infrastructural aspects of thermocouple principles,

measurement, and test methods as well as calibration 

services. This study, published in July 1997, conservatively 

estimated the social rate of return to be 32 %, and a benefit-to-cost ratio of 2.95. 

Now available online at 

http://srdata.nist.gov/its90/main/

Recent Developments: Currently, the ITS-90 relies on the fundamentally more accurate high

temperature platinum resistance thermometer to define the temperature scale in the range of

631 °C to 1064 °C. In this range, prior to the introduction of the ITS-90, less accurate type S 

(platinum-rhodium vs. platinum) thermocouples were used to define the temperature scale. Work

at NIST based on the new scale definition led to new reference functions with improved accuracy 

for both type S and R thermocouples. Even more importantly, the new definition of the ITS-90

enabled the determination at NIST of highly accurate reference functions for pure element thermocouples, such as gold versus platinum and platinum versus palladium. In the range from 0 °C to

1000 °C, gold versus platinum thermocouples provide an order of magnitude more accuracy than the

previous type S thermocouples commonly used as 

reference standards in industry. 

Based on the NIST research, gold versus 

platinum thermocouples are now readily

available to the user community from 

several U.S. manufacturers. 

 

The next sections of this Report will delve into each of CSTL’s 13 programs individually, highlighting major activities and accomplishments in these areas for FY2003. Following the highlights of 

the 10 customer-focused programs, this Report addresses the three goal-aligned programs. Most 

of the details of the projects listed in the Measurement Standards, and Data and Informatics Sections are presented in the context of their impacted industry sector, and a reference is supplied to

provide a more coherent view. CSTL, however, also works on the cutting-edge of metrology and

develops tools to anticipate next generation needs. These tools are not necessarily identified

with a particular industry sector, and therefore are presented here in the program title: Technologies for Future Measurements and Standards, which reflects CSTL’s third strategic goal. 

Technical details of these activities can be found on CSTL’s website or by contacting the principle

investigators listed in the Report. 

http://www.cstl.nist.gov/

13
14
B. CSTL Programs 

Pharmaceuticals and 

Automotive and Aerospace Biomaterials Biomanufacturing 

Energy and

Environmental 

Technologies 

Chemical and 

Allied Products 

Food and Nutrition Health and Medical 

Technologies 

Forensics and 

Homeland Security

Microelectronics 

Measurement Standards

Data and Informatics

Industrial and

Analytical 

Instruments and

Services

Technologies for Future 

Measurements and Standards 

15
16
1. Automotive and Aerospace (Transportation) 

CSTL provides standards 

for emissions testing and 

flow measurements to 

help the transportation

industry meet regulatory 

requirements. CSTL also 

works with the industry to 

provide physical and 

chemical property data that can be used in sophisticated computer models for next generation

combustion engine design, and provides a new generation of chemical standards and reference

data to support advanced materials such as super alloys for high technology jet engines. 

When California Low Emissions 

Vehicle (LEV11) regulation is fully

implemented in 2010 it is estimated 

that smog-forming emissions in the 

Los Angeles area will be reduced by

57 tons per day, while the statewide 

reduction will be 155 tons per day.

New Gas Standards for Calibrating Instrumentation Used for Measuring Emissions from 

Next Generation Low Emission Vehicles 

W.J. Thorn III and W.D. Dorko (839) 

Stakeholders in the American Industry-Government 

Emissions Research (AIGER) group are working

together to facilitate the automobile industry meeting

more stringent 2003 Federal Tier II and California 

LEV II emission regulations. AIGER members include 

the U.S. EPA, California Air Resources Board

(CARB), General Motors, Ford, and DaimlerChrysler. NIST and the U.S. Motor Vehicles 

Manufacturers have worked together since 1975 to 

develop sixty gaseous Standard

Reference Materials, which are the 

Nation’s benchmarks against which all 

U.S. EPA mandated fuel economy and mobile source emission measurements must be traceable

by federal law. In 1998, NIST worked with a Specialty Gas contractor to blend two cylinders each

of five low NO standards at concentrations ranging from 

of 0.5 μmol/mol to 1.25 μmol/mol. The ten new 

standards were prepared employing a NIST-developed

cylinder pretreatment, a NIST SRM 2629a as the parent

NO source and a diluent balance gas of specially

scrubbed (active metal getter) nitrogen (O2 < 2 

nmol/mol). 

CSTL researcher developed a 

novel pretreatment process that 

passivates gas cylinder walls 

allowing the long-term storage of 

low level NO gas permitting the 

commercial production of this 

high priority standard. 

Newer vehicles produce lower levels of these pollutants 

because their engines employ fuel injection whose airto-fuel ratio is optimized by an on-board computer, and 

their emissions are further reduced by more efficient

catalytic converters. During testing the exhaust levels 

are diluted by the use of constant volume sampling bags

or by new mini-diluter technology. AIGER stakeholders 

have identified the need for significa

standards containing carbon monoxide, hydrocarbons and nitric oxide; with

the completion of SRM 2737 (0.5 μmol/mol) and SRM 2738 (1.0 μmol/mol) 

nitric oxide in nitrogen being their highest priority. NIST responded with

thirteen cylinders at nominally the same concentration levels of SRM 2737 

and SRM 2738. These were recently analyzed by NIST and returned to 

The low NO standards have been 

analyzed periodically against NIST 

dynamic permeation standards and 

have exhibited excellent NO conc. 

stability (<2 % relative) for approx. 

five years. See example below:

Stability of Low NO Standard 

(Nominal Concentration 

0.500 μmol/mol) 

Year Sample 1 Sample 2 

1999 0.499 0.507 

2000 0.502 0.512 

2001 0.500 0.514 

2002 0.501 0.516 

2003 0.491 0.506 

ntly lower NIST gas 

17 
AIGER members for their interim use − until the new SRMs become available in March 2004. 

NIST assigned NO and NOX values to 

the NIST low NO standards by two

different methods. Shown is the NO 

calibration curve in which the 

assigned NO concentration is plotted 

on the x-axis, and the ratio of the 

measured value to the SRM 2738 lot 

standard is plotted on the y-axis. All

candidate SRM mixtures at 0.5

μmol/mol and 1.0 μmol/mol were

analyzed in December, 2002, which

was sixteen months after blending. All

cylinders are stable and all contain 0.2

μmol/mol nitrogen dioxide. 

NIST SRM 2738 NO Chemiluminescence 

Calibration Line

0.300

0.500

0.700

0.900

1.100

1.300

0.300 0.500 0.700 0.900 1.100 1.300

NO Concentration - μmol/mol

SRM 2738 Lot 

Standard

The project has technically resolved NIST’s low NO SRM stability problems, provided AIGER 

members with interim standards to work with, and will 

soon provide much needed lower concentration NIST

certified NO SRMs in the near future; and thus help

facilitate vehicle manufacturer’s in meeting U.S. EPA and

CARB’s current and future lower emission regulations. In 

addition, AIGER and NIST are discussing technical 

issues to upgrade the NTRM protocol to meet mobile 

source accuracy and stability criteria for emission

measurements. The NTRM “prime” (NTRM’) addendum 

to NIST SP 260-126 has been submitted to AIGER for comment. 

A rollout of the new NTRM’ gas 

standards are scheduled at a 

meeting between AIGER, specialty

gas companies and NIST to be 

held in parallel with the March 

2004 Pittsburgh Conference 

meeting in Chicago. 

Fourier-Transform Microwave Spectroscopy (FTMW) Critically Evaluated for Real-time 

Measurements of Automotive Exhaust Emissions 

R.R. Bousquet and P.M. Chu (839) 

Fourier-transform microwave (FTMW) 

spectroscopy is being investigated as a means 

for quantitative real-time measurements of 

automobile exhaust emissions. The principal 

goal of the present program is to critically

evaluate the technique’s potential as a

sensitive, reliable, and robust tool for

quantitative measurements of trace gases in 

complex gaseous mixtures for real-time 

analysis of automobile exhaust emissions,

environmental monitoring, process control, and homeland security applications. 

FTMW spectroscopy combines the high 

spectral resolution of microwave 

spectroscopy and the high sensitivity 

of molecular beam and cavity-enhanced 

methods creating a technique that can 

provide unambiguous identification of 

vapor phase analytes that possess 

permanent electric dipole moments.

Current work has focused on improving the 

sensitivity of the detection system and

characterizing the effects of species such as

water and carbon dioxide present in the sample

gas. Continued efforts to improve the signal 

stability and reproducibility pushed the stability to

approximately 1 % over 24 h. Further development continues in a number of areas including: 

use of low noise preamplifiers in the initial signal processing, implementation of a dual phase 

In FY 03, additional effort was focused on 

characterizing the factors that determine 

the overall sensitivity of the detection 

system and upgrading the software and 

hardware to enhance the signal to noise. 

18 
modulation/detection system, improvements

of the cavity mode quality, and addition of 

adjustable antenna for optimization of the 

microwave radiation transmission at specific 

analyte transition frequencies. Finally, 

detection limits ranging from 0.1 μmol/mol to

10 μmol/mol have been realized for several

analytes in pure nitrogen matrices.

Furthermore, minimal changes in the

detection limits were observed for the

addition of ambient levels of oxygen, carbon

dioxide and water to the sample matrix

suggesting that this is a viable technique for 

real-time ambient monitoring. 

Accurate quantitative measurements of trace 

species in bulk gases are becoming more

vital to both science and industry with 

detection requirements to the low part per billion regions and beyond. Analytical methods that 

exhibit high chemical selectivity and 

sensitivity are particularly advantageous 

for the analysis of complex matrices.

Through this research we hope to 

demonstrate the potential of FTMW

spectroscopy to help meet these 

measurement needs. 

Schematic diagram of FTMW system, 

and sample of spectral output. 

Improvements planned for FY 2004 should push the 

detection limits of the FTMW spectrometer to the 

nmol/mol to pmol/mol region. Additionally, to 

facilitate the deployment of the FTMW spectrometer 

in field and laboratory applications, higher-pressure 

measurements will be investigated. 

Properties of Rocket Propellant (RP-1) 

J. W. Magee, D. G. Friend, T. J Bruno, M. L. Huber, E. W. Lemmon, A. Laesecke, R. A. 

Perkins, and J. A. Widegren (838); I. M. Abdulagatov (Russian Academy of Sciences) 

The National Aeronautics and Space

Administration (NASA) has, for decades, used a 

kerosene blend, designated RP-1, as a rocket 

propellant. Presently, NASA and its contractors are 

designing advanced rocket engines that will 

combust RP-1 fuel. While reliable thermophysical 

properties are essential for this purpose, presentday models are either based on a pure component

surrogate compound (dodecane) or on estimation 

methods that are grounded in very limited data. To

address the issues surrounding the accuracy of

thermophysical properties of RP-1, new 

measurements and models are needed. 

CSTL researchers are supplying NASA 

with validated property information at 

high temperatures and pressures for 

the development of a Rocket Engine 

Prototype planned for future launch 

vehicles. 

Prior to the work reported here, published data were limited to near atmospheric pressure and

temperatures to less than about 373 K. A measurement program is underway that is intended to

provide high quality data in order to support the advanced model development. Measurements of

chemical composition, stability, density, heat capacity, viscosity and thermal conductivity are

being conducted in a range of temperatures up to 700 K and pressures to 60 MPa. The first

compositional studies, based largely on a gas chromatography - mass spectrometry (GC-MS) 

19 
method, have identified key components and classes of chemicals found in a sample of RP-1. 

Initial measurements of density and viscosity have been completed, and advanced models,

based on both published data and these new measurements, are under development. 

A GC-MS method was used to chemically characterize a sample of RP-1 supplied by NASA. As

expected, the ion chromatogram showed that RP-1 is a complex liquid fuel that consists of

significantly more than 100 components. When the best resolved chromatogram peaks were

individually integrated and interpreted, a report was 

prepared that identifies the key constituents of this

mixture. From this report, an initial slate of compounds

has been selected for a surrogate mixture to be used for 

modeling thermophysical properties. The selection was 

based on the predominance in RP-1 and the variety of 

chemical structures to represent key classes of compounds identified in the complex fuel. An

experimental program was initiated to measure important thermodynamic (density and heat

capacity) and transport (viscosity and thermal conductivity) properties of RP-1 in ranges of 

temperature from 300 K to 700 K and pressure from 0.1 MPa to 60 MPa. The first measurements

reported were the density and viscosity. Modeling studies have concentrated on the pure 

compounds comprising the surrogate mixture. Short equations of state and models for the 

transport properties have been developed for each of these constituents of RP-1, and a

preliminary Helmholtz energy mixture model has been developed that is based using a set of 

predicted interaction parameters. Similarly, a preliminary mixture model has been nearly 

completed for the transport properties of RP-1. 

In FY 03, density and viscosity

measurements have been reported 

in ranges of temperature from 300 K 

to 700 K and pressures from 0.1 

MPa to 60 MPa. 

Properties and Processes for Cryogenic Refrigeration 

R. Radebaugh, P. Bradley, and M. Lewis (838); J. Gary and A. O'Gallagher (ITL); E. Luo, 

(Chinese Academy of Sciences). 

Cryocoolers are required for many technology areas, including the cooling of infrared sensors for 

surveillance and atmospheric studies, the

cooling of superconducting electronics,

magnets, and power systems, the cooling

of cryopumps for clean vacuums in

semiconductor fabrication processes, the 

liquefaction of industrial gas, and many 

other existing and potential applications. 

This research aims to develop

measurement and modeling techniques 

for evaluating and improving performance 

of cryocoolers and their components; to develop 

new and improved refrigeration and heat transfer 

processes below about 230 K; and to provide an 

electronic database on cryogenic material 

properties. 

CSTL researchers have developed test 

measurements that led to obtaining 

temperatures of about 17 K in a small twostage pulse tube refrigerator that we designed 

to achieve 15 K. This prototype is the first 

step in the development of a system for 

subcooling liquid hydrogen to 15 K for use in 

the next generation space vehicle. 

CSTL researchers worked with Sierra Lobo on the refrigeration system for

supercooling liquid hydrogen for the NASA’s next generation space vehicle. We 

collaborated with scientists at Sierra Lobo, a small company doing research in 

cyrogenic technologies, to investigate the technology of densifying hydrogen 

Next steps would involve scaling the overall system to much larger sizes if NASA chooses to

develop this approach. 

20 
With a medical device company, we completed measurements on the performance of a simple 

pulse tube refrigerator for the cooling of dermatological pens to about 183 K. Such temperatures 

are higher than typical operating temperature of pulse tube refrigerators, and although our models 

predicted the performance well, issues with the compressor have hampered some necessary 

measurements. 

We have carried out measurements of the complex 

fluid dynamic impedance of small inertance tubes and

compared them with two types of models. Our 

measurements revealed that typical laminar flow 

elements have large errors in the measurement of 

oscillating flows, whereas hot-wire anemometers do

not. This year, a large (500 W at 30 K) prototype

pulse tube neon liquefier was developed, and

extensive tests have now been completed to measure

losses in both the regenerator and the pulse tube. The

pulse tube effectiveness was found to be even better

than expected, but losses in the regenerator were

higher than expected. Measurements indicated that temperature non-uniformities exist which

may be a result of non-uniform flow; we have not yet been able to pinpoint the reason for such

behavior. Solving this problem is very important for large pulse tube refrigerators being

considered for cooling of power applications of superconductors (motors, generators, 

transformers, and fault current limiters). 

The use of these technologies has 

been hampered because of problems 

with existing cryocoolers, including 

short lifetimes, inefficiency, high cost, 

and excessive vibration. Metrology

needs to be developed to characterize 

losses, and models are needed to 

optimize the design of such systems. 

Material properties at cryogenic 

temperatures are required for the 

design of cryogenic equipment. 

Dissemination of data and information: 

• Papers were presented at the Cryogenic 

Engineering Conference on the results of 

our measurements and modeling of 

inertance tube behavior. 

• A book chapter on “Refrigeration for 

Superconductors” has been developed 

for a special IEEE Proceedings on 

Electronic and Large Scale Applications 

of Superconductivity, covering the 

fundamentals of refrigeration for both

small and large superconducting 

systems. 

• Database on the properties of cryogenic 

materials to be expanded.

In FY 03, we have provided a wide range of

industries with new tools to improve the

performance of cryocoolers, particularly those

operating at temperatures below about 50 K and 

above 150 K. Our model for inertance tubes led 

to the publishing of design curves for their 

optimization, and the model results in more 

efficient cryocoolers. Our work on two-stage 

pulse tubes at 30 K and below will aid the

superconductor industry in the development of 

superconducting systems that can be marketed 

to a larger customer base and improve the

electrical infrastructure of the U.S. Our materials 

properties database is now receiving

considerable attention and is useful to all 

industries dealing with cryogenic temperatures. 

An expanding customer base is requiring our 

models, data, and technical expertise in the

development of cryocoolers for an ever-increasing range of applications. To improve efficiency of

these coolers, we are continuing the development of a test apparatus to measure nearly all types 

of losses in cryocoolers and to measure how they are influenced by various parameters. 

21 
Benchmark Data on Liquid Fire Suppressants for Use in Aircraft 

C. Presser and B. Johnson (836); C.T. Avedisian (Cornell University); G. Papadopoulos 

(Dantec Dynamics); J. Hewson (Sandia National Laboratory); D. Keyser (NavAir); P. 

Disimile and J. Tucker (46th Air Force Test Wing) 

The new generation of non-ozone-depleting 

Halon fire suppressants include chemicals that

have higher boiling point temperatures (i.e., >330 

K) and that exist in the liquid phase under highpressure release or in ambient conditions.

Release of these agents in a confined and

cluttered space such as aircraft dry bays and 

nacelles results in the dispersal of droplets that either travel 

along ballistic trajectories, move with the convecting flow, or

impact upon nearby solid obstacles. An accurate

representation of droplet transport is crucial for the 

numerical modeling of fire suppression in obstructed spaces

in aircraft. 

An accurate representation of droplet 

transport is important in predicting the 

success of fire suppression in 

obstructed spaces in aircraft using fire 

suppressant that have high boiling 

points. 

To better understand the physics of droplet 

transport around and behind solid objects, an

experimental arrangement was set up to impose 

controlled grid-generated turbulence on the air

stream. Experimental results from this facility 

provide new experimental data for a wellcharacterized, homogenous, droplet-laden 

turbulent flow field around prescribed obstacles to provide data for flow model validation. 

This project will provide a parametric data 

set to validate the VULCAN computational 

fluid dynamics fire physics code for sprayclutter interactive environments, which is 

under development at Sandia National 

Laboratories.

The agents considered in the investigation were 3MTM fire protection fluorocarbons 

HFE-7100 and HFE-7000 with

boiling points of 334 K and 307 K,

respectively, in addition to water.

Phase Doppler measurements were used to 

obtain droplet size and velocity distributions in

a droplet-laden homogenous turbulent flow 

field around a horizontal, 32 mm diameter 

cylinder serving as an obstacle. These 

measurements help elucidate the effects of 

different physical properties of the agent on 

droplet transport, as well as the effect of 

droplet size on impact and coating of the

cylinder surface, rebound into the free 

stream, vaporization near the cylinder as it is 

heated, and entrainment behind the cylinder. 

The results indicated that, unlike water,

HFE7000 and HFE7100 vaporized readily 

without any droplet accumulation and dripping 

from the cylinder. Droplet size is correlated 

with boiling point. Downstream in the wake of the cylinder, a distribution of smaller size droplets 

(generally, less than 30 μm) were entrained in the recirculation zone. Comparison of results for 

the unheated and heated cylinder with water found that near the heated cylinder surface, droplet

vaporization resulted in smaller mean droplet sizes, relative to the unheated case. 

Apparatus showing phase Doppler 

transmitter (right) and horizontal cylinder 

(lower left) within Plexiglas® chamber.

22 
2. Biomaterials 

The biotechnology industry is making significant advances in the areas of 

biomolecular research for use in new environmentally friendly materials and

process technologies. CSTL supports these efforts by developing 

measurement methods, standards and tools 

for quality control, as well as standards for 

calibration of specialized bio-based 

equipment. In addition, advances in tissue 

engineering continue to grow rapidly requiring standards to assure 

the integrity of tissue-engineered products during development,

storage or shipment. 

The total market for the 

regeneration and repair 

of tissues and organs is 

estimated to be $25 

billion worldwide. 

Desiccation Tolerance of Genomic DNA of the Cyanobacterium Nostoc Commune 

H. Rodriguez (831), M. Birincioglu (Guest Researcher); P. Jaruga (CARB, UMBI); B. Shirkey, 

S.C. Smith, D.J. Wright, and M. Potts (Virginia Tech Center for Genomics and Virginia 

Tech, Blacksburg, VA) 

Desiccation is likely to have been an important determinant 

in the evolution of primitive cells. Today, desiccation 

remains the most acute environmental stress suffered by

living cells. It is known that some cells remain desiccated

for extended periods of time (decades or more), however, 

the mechanisms that enable a cell to survive as it enters 

and leaves the air-dried state are unknown. The

Cyanobacteria was selected for this work since it is

ecologically important as well as a phylogenetically diverse

assemblage of prokaryotes, thus offering an excellent

model system for the study of nitrogen fixation, photosynthesis, and cell differentiation. Nostoc 

commune has a cosmopolitan distribution and is of particular interest in that it has a marked 

capacity for desiccation tolerance. 

Water is indispensable for 

the maintenance of cell 

integrity and function. The 

removal of water from a cell 

causes such severe 

perturbation that the cell may 

die within seconds of its 

exposure to the atmosphere. 

This work demonstrated the stability of desiccated

DNA in vivo. Key physiological processes and 

metabolic networks that contribute to desiccation

tolerance using functional genomics and 

proteomics have been identified. This work is 

complimentary to the ongoing research in CSTL in

the field of biomarkers of tissue engineering. It

broadens the work at NIST by directly applying the biomarkers 

developed for the field of tissue engineering to the field of cell 

desiccation tolerance and cryopreservation. Understanding these

processes will aid the development of better long-term storage methods 

of tissue-engineered medical products. 

Cyanobacteria, 

Nostoc commune

DNA from Nostoc commune

ENG1996 colonies desiccated 

for four years at time of assay or 

rehydrated with distilled water 

for time periods before isolation 

of the DNA for RAPD-PCR 

assay. Lanes from left to right: 

from dry to rehydration for 5 

min, 30 min, 1 h, 3 h, 24 h. 

Complete technical results are reported in 

Nucleic Acids Research, 2003 vol 31, No 

12. 

27 
Comparison of Reagents for Shape Analysis of Fixed Cells by Automated Fluorescence 

Microscopy

J.T. Elliott, A. Tona, and A.L. Plant (831) 

Morphology analysis of cells in culture is commonly employed

by pathologist and cell biologist to characterize cell behavior. 

For example, cell morphology can be a marker for cell 

phenotype or aberrant cell behavior. In addition, cell size and

shape have been implicated as potentiators of intracellular 

signaling events. Automated microscopy and image analysis 

can provide quantitative information about the size and shape 

of cultured cells, but it requires that the edge of a cell can be

clearly identified. Generating adequate contrast at the edge of 

thin well-spread cells cultured on solid substrates can be 

challenging with traditional staining techniques. Therefore we 

conducted a comparative analysis of six fluorescein-based

probes for their effectiveness as whole cell stains for automated

morphology analysis of fixed cells. The identification of a high-contrast and robust staining

procedure for fixed cells will be useful to any research groups that use cell morphology to address 

how culture conditions, extracellular matrix modification and biomaterial substrates influence

cellular behavior. 

CSTL researchers have 

identified a fluorescent 

cell stain to generate high 

intensity contrast at the 

periphery of fixed cells, 

thus improving the quality

of cell morphology 

measurements made by 

automated microscopy 

and image analysis 

routines. 

The results of this study indicate that formaldehyde fixed

cells stained with fluorescein-5-maleimide exhibited the

highest contrast at the cell edge and required 25-fold

shorter exposure times than cells stained with 5-

chloromethyl fluorescein diacetate (CMFDA, Cell 

Tracker Green), a commercially available whole cell 

stain. In addition, the average area determination of 

cells stained with fluorescein-5-maleimide was less 

sensitive to changes in the image analysis parameters 

(threshold value) that are used to identify cell objects. 

The stain described here is used 

routinely in our laboratory for 

morphological evaluation of 

cultured cells on biomaterials. We 

have recently begun to combine this 

stain with other cell stains to 

improve the level of quantitative 

information that can be gathered 

from cells in culture. 

Figure to left shows vascular smooth 

muscle cells stained with fluorescein-5-

maleimide, the brightest stain identified in 

this study. The high contrast exhibited at

the periphery of the stained cells facilitates 

automated object detection with image 

analysis software. Figure to right shows the

outline of a cell that was identified by

automated image analysis. 

 

 

28 
Characterization and Control of Gel-Fluid Phase Transitions in Supported Phospholipid 

Monolayer Films 

L.J. Richter (837); C.S.-C. Yang, J.C. Stephenson, and K.A. Briggman (PL) 

 

The physical structure (gel or fluid phase) of lipid domains in membranes influences cellular 

signaling and other biological function, and hence 

must be characterized for both model and biological 

systems. All cells are surrounded by plasma

membranes based on lipid bilayers. There is 

increasing evidence that the physical structure of the 

lipid membrane has important physiological 

implications. Lipid microdomains consisting of high 

phase transition temperature lipids and cholesterol 

have been implicated in the function of signaling 

proteins. Conventional calorimetery cannot be done

on planar model systems, due to the low surface area. Thus, very little is known of the thermal 

phase behavior of lipids in supported structures. In order to address this knowledge gap, CSTL

researchers are validating optical techniques for the characterization of the thermal phase of 

supported lipid constructs.

Hydrated supported phospholipid 

structures (Langmuir-Blodgett films, 

supported bilayers, etc.) have been 

widely studied as model systems for

biological membranes. Supported 

monolayers and bilayers may also 

be used as platforms for novel 

biosensors and biomaterials. 

Vibrationally-resonant sum frequency generation (VRSFG) is a non-linear optical probe uniquely suited for

the in-situ characterization of planar interfaces, as the

signal is symmetry forbidden in centrosymmetric 

media. This selection rule also makes VR-SFG ideally 

suited to the study of the gel-fluid phase transition in

phospholipids, as the methylene CH stretch spectral

features in the gel phase are forbidden by the all-trans 

symmetry of the alkane chains, while these features 

are allowed in the disordered chain fluid phase. 

In a typical structure of a hybrid monolayer (HBM) a

lipid monolayer formed on a hydrophobic film on a 

solid substrate. Independent control over both the lipid

layer and the hydrophobic film may be used to

optimize the HBM for specific applications. The VRSFG spectra of HBMs formed with two different

phospholipids are shown in the figure: Dipalmitoyl-snglycero-3-phosphocholine (DPPC) and Dimyristoyl-snglycero-3-phosphocholine (DMPC), with 14 and 12

methylene groups per alkane chain, respectively. The 

lipid chains were perdeuterated to provide contrast 

against the protonated support. The top graph depicts 

the ratio of the thermal phase dependent methylene

CD stretch feature to an internal standard, the CD 

stretch signal from the terminal methyl groups, for the 

phospholipids on a well ordered octadecanethiol 

(ODT) on gold support. The inflection in the step-like 

increase in the ratio indicates the temperature of the gel-fluid phase transition TM in the two films. 

As expected, the longer chain DPPC has a higher TM than the shorter DMPC. 

Scaled fluid phase spectral intensity

for HBMs formed with DMPC (red) and 

DPPC (blue). Vertical Lines mark the 

TM for unsupported vesicles. (Top) 

HBM support is a pure ODT or TL film. 

Arrows indicate shift in TM; (Below)

HBM support is mixed TL/MCH film. 

TM HBM ≈ TM vesicles. 

29 
The TM of HBMs formed on the ODT support is 

uniformly shifted by about 10 °C to higher

temperature from the TM of vesicles. This 

perturbation of TM is also observed for HBMs on a

support formed from a well-ordered thiolated lipid

(TL) film of Dipalmitoyl-sn-glycero-3-phosphothioethanol. It was discovered (data in graph B) that 

TM can be controlled by selectively incorporating a

small hydrophilic molecule, mercaptohexanol (MCH), 

into the TL film, introducing slight structural disorder 

and weak polar forces. HBMs formed on supports fabricated with a 1:2 mole ratio of MCH to TL in

the film-forming solution have TM essentially identical to that of vesicles. 

VR-SFG has been established as a 

diagnostic for the thermal phase of 

supported bilayers. It was discovered 

that TM for the most commonly formed 

HBMs is significantly different from that 

of bilayers in vesicles. This change in

TM could affect the biomimetic 

characteristics of HBMs. A procedure

for controlling TM via modification of

support was developed. 

Next Steps: The ability of VR-SFG to characterize the thermal phase of supported lipid films and 

the ability to control the phase transition via modifications of the support will be used to study the 

influence of lipid phase on the insertion and structure of small membrane bound proteins and 

peptides. 

30 
3. Pharmaceuticals and Biomanufacturing 

CSTL provides a breadth of resources that support the pharmaceutical 

industry including reference data as well as 

artifact standards such as optical filters and

fluorescence standards used for instrument 

calibration. CSTL measurements and 

standards facilitate the drug discovery 

process, help optimize production of new 

pharmaceuticals, and ensure quality control in

manufacturing processes. In addition, CSTL’s 

fundamental work in enzyme characterization promotes the transition to

biomanufacturing leading to more environmentally sustainable

manufacturing.

Increased demand for 

new products has 

replaced price as the 

primary force in the U.S. 

pharmaceutical 

industry’s overall 

growth. 

Bioseparations - Separation of Different Topological Forms of Circular DNA 

K.D. Cole (831) 

The DNA from many microorganisms and cellular organelles is circular. Plasmids are important 

examples of circular DNA that are widely used to clone

genes and express foreign proteins. We are studying the

separation of circular DNA to improve methods for the

analysis and the isolation of large amounts of plasmid

DNA. Circular DNA can be selectively retained in gels by 

electric fields by a process termed electrophoretic 

trapping. Our efforts have been to characterize the

process, develop models, and attempt to exploit

electrophoretic trapping for improved analytical and

preparative methods for circular DNA. The migration of 

circular DNA can be completely arrested in gels when the 

electric field strength reaches a critical value. 

Improved methods for the 

production, characterization, 

and purification of circular 

DNA are needed to meet the 

needs of the pharmaceutical, 

biotechnology, and molecular 

biology communities. 

31

CSTL researchers have studied the effects of the gel structure

and the topological form of the DNA on trapping. The 

concentration of polymer (agarose) used to form the gel has a

large effect on the formation of the “traps” for circular DNA. 

The electric field strength 

for the onset of trapping,

the apparent trap density, and the release characteristics of

circles from traps were studied using both direct current and 

field inversion gel electrophoresis (FIGE) experiments. The 

average distances before 13 kbp (kilobase pairs) open circles

were trapped were ≈ 80 μm in 0.25 % gels, 180 μm in 1 % 

gels, and greater than 500 μm in 2.5 % gels. The higher trap 

density and longer trap length in low concentration gels explains the experimental observations

that under some conditions DNA circles migrate faster in higher concentration gels compared to

their migration in lower concentration gels. Lower concentration gels had a higher density of

traps and the traps were more heterogeneous when compared to traps formed in gels formed 

with higher concentrations of agarose. 

The factors that determine 

the formation of the 

electrophoretic traps in gels 

are not well understood. A 

better understanding of 

these factors would allow us

to control their formation

and to design better gels for 

the selective separation of 

circular DNA. 

 “The influence of agarose 

concentration in gels on the 

electrophoretic trapping of 

circular DNA,” Cole, K. D.; 

Åkerman, B., Separation Sci. 

Technol. 38, 2121-2136 (2003). 

An apparatus was designed for the electrophoretic capture and recovery of circular DNA in thin 

layers (membranes). Rapid separations were done by the use of a low conductivity buffer and 
high electric field strengths. Two 

methods that specifically retain circular

DNA in the membranes are 

demonstrated using the supercoiled and 

open circular forms of two plasmids with

sizes of 4.4 kbp and 13 kbp.

Electrophoretic trapping (by an

impalement mechanism) in agarose gelfilled membranes used electric field 

strength to immobilize circular DNA in the

membranes. These membranes and 

methods should be scaleable to process 

large number of samples or to prepare 

larger amounts of pure circular DNA by 

increasing the area of the membranes. 

The apparatus facilitates the rapid

screening of a large number of trapping 

materials and methods for recovery of

DNA. 

Schematic diagrams of the apparatus used for 

electrophoretic capture in thin layers and gel 

electrophoresis of DNA purified using the apparatus 

showing the location of supercoiled (SC) and open

circular (OC) plasmid DNA. The recovered plasmid

DNA is free of genomic (host) DNA. 

These studies permit the more rational design of gels 

for the separation of circular DNA. The gels can be

designed or “tuned” for a particular analytical or

preparative separation and thus give an improved 

separation of the 

circular DNA. We

plan to extend this research to use fluorescence microscopy in

order to observe individual circular DNA molecules, tagged with

fluorescent dyes, as they separate in gels. This will provide

insight into the mechanism of separation and thus allow us to 

design improved separations. 

 “An apparatus for electro-phoretic 

trapping of circular DNA in thin 

layers,” Cole, K. D. Biotechnol. 

Applied Biochem. 37, 251-257 (2003). 

Both the data and the 

apparatus can be used by

researchers to prepare 

highly pure circular DNA in a 

matter of minutes.

Develop Imaging Tools to Characterize Fluorescence from Fluorophores Immobilized on 

Surfaces 

A. Gaigalas, K.D. Cole, and L. Wang (831) 

A large variety of new fluorescent reagents have

been developed for the measurement of biological 

targets either in solution or on surfaces. These 

fluorescent dyes have to a large extent replaced

the use of more dangerous and less convenient

radioisotopes. However, the use of fluorescent

reagents to detect biological targets on surfaces is 

not without their drawbacks, including the

photobleaching of the fluorophore, fluorescence 

quenching of the light due to interactions in the 

microscopic environment on the surface, and background subtraction. The research described

here aims to improve the limits of detection of targets and to increase the quality of the data

obtained. 

Imaging of fluorescent signals from 

surfaces has a number of important 

applications in biotechnology, 

including the detection of cell types, 

viruses, and measurement of target 

molecules. Advances in genomics 

and proteomics have made larger 

scale screening techniques possible. 

32
33

40μm 

Open circles trapped

in gel

electric

field 

I

chr

CSTL researchers are measuring the process of photobleaching in solution and the effect of

protective additives on the rates of bleaching. A fluorescent 

microscope has been set 

up to image targets 

including DNA molecules,

viruses, bacteria, and 

microarrays. We are 

starting measurements of

these targets and the 

evaluation of the images 

using commercial software

programs. We 

are in the 

process of 

improving the microscope to improve the quality of the 

images obtained. These preliminary experiments have

also indicated the importance of the sample preparation (labeling with the fluorophore) and the

surface environment on the quality of the measurements obtained. 

Effective addictives help to prevent 

fluorophores from photodegrading 

and photobleaching. This serves 

to increase the limit of detection 

and the overall contrast to the 

auto-fluorescence background. In 

turn, this enhances and makes 

possible applications such as virus 

and bacteria counting and the 

imaging of individual microarray

spots. maging of bacterial artificial 

omosomes (220,000 base pairs)

during agarose gel electrophoresis.

We plan to continue measurements of

photobleaching for different fluorescent 

reagents and the effect of stabilizing 

additives. The effect of different 

surfaces and substrates on fluorescence

quenching of fluorophores and

background will be examined. 

“A frequency-domain technique for studying 

photodegradation process of fluorophores,” 

Gaigalas, A. K.; Wang, L.; Vogt, R. F., Photochem. 

Photobiol. 76, 22-28 (2002); 

“Photodegradation of fluorescein in solutions 

containing n-propyl gallate,” Gaigalas, A. K.; Wang, 

L.; Cole, K. D.; Humphries, E., in review. 

Enzyme-Catalyzed Synthesis of Optically Active Drug Intermediate Compounds Using 

Supercritical Fluids as the Solvent 

Y.B. Tewari (831); K.W. Phinney (839); T. Ihara (AIST, Japan) 

Recently, supercritical carbon dioxide (SCCO2) has been used

as an alternative solvent for biochemical reactions and has 

been shown to have several advantages over commonly used 

organic solvents. Specifically, 

carbon dioxide is non-toxic, 

non-flammable, available in 

abundance, and is

environmentally friendly and

recyclable. The low viscosity 

and high diffusivity of SCCO2 

serve to provide favorable mass 

transfer properties. In this work, 

a reaction vessel was designed, 

fabricated, and tested to study enzyme-catalyzed reactions in

SCCO2 media. The equilibrium constants of the lipase-catalyzed 

transesterification reaction (benzyl alcohol + butyl acetate = benzyl 

acetate + 1-butanol) were studied in SCCO2 . The equilibrium

constant of this reaction in SCCO2 is similar to those obtained in

In the pharmaceutical 

industry, enzyme-catalyzed 

reactions are used for the 

synthesis of chiral drug 

intermediates. Because 

these reactions are 

generally carried out in 

organic solvents, the 

generation of large volumes 

of organic waste is of 

concern. 

The enzyme used in this 

study was a lipase from 

Candida antarctica. 

(Structure from PDB) 
n-hexane, toluene, and other non-aqueous solvents. These results also show that the rate of

reaction in SCCO2 media is higher than those in organic 

solvents. 

This is the first equilibrium study that has been performed in

supercritical carbon dioxide. We have demonstrated that 

supercritical carbon dioxide provides kinetic and 

environmentally friendly advantages for carrying out 

enzyme-catalyzed reactions. This work provides 

comparisons between enzyme-catalyzed reactions in 

traditional non-aqueous solvent systems and in supercritical 

carbon dioxide. The data will facilitate implementation of 

supercritical carbon dioxide as a solvent for biocatalysis in

the biotechnology and pharmaceutical industries. 

Alternatives to organic solvents are important for reducing 

the levels of residual solvents in pharmaceutical products 

and for reducing the environmental burden resulting from 

solvent waste.

Reaction quotient vs time in 

different solvent systems. The 

solvents used were n-hexane (Ο), 

toluene (), carbon dioxide (×), and 

the system without solvent (). 

Studies of additional enzyme-catalyzed reactions in

supercritical carbon dioxide are planned. These investigations will focus on reaction systems of

industrial significance and relevance. 

“A Thermodynamic and Time Course Study of the Lipase-Catalyzed Transesterification of 

Benzyl Alcohol and Butyl Acetate in Supercritical Carbon Dioxide Media,” Tewari, Y. B.; Ihara, 

T.; Phinney, K. W.; Mayhew, M. P., submitted to J. Mol. Catal. B: Enzymatic. 

Critical Evaluation of Capillary Electrophoresis for the Determination of Enantiomeric 

Composition of Ephedra Alkaloids 

K.W. Phinney and L.C. Sander (839); T. Ihara (AIST, Japan) 

34

Ephedrine and pseudoephedrine are two of the 

major alkaloids found in ephedra, but these 

compounds are also found in traditional 

prescription and over-the-counter medications.

Both compounds exist as pairs of enantiomers, but 

only (-)-ephedrine and (+)-pseudoephedrine are 

found in ephedra plants. Detection 

of (+)-ephedrine, (±)-ephedrine,

or (-)-pseudoephedrine in a

product containing ephedra would suggest that the content had been 

altered through the addition of synthetic alkaloids. Hence, the 

enantiomeric composition of these compounds is important in verifying the 

natural (plant) origin of the 

alkaloids. 

Dietary supplements containing 

ephedra have generated considerable 

interest because of their proposed 

ability to promote weight loss and 

increase athletic performance. 

However, ephedra has also been 

associated with adverse health 

effects. 

Ephedra plant 

The high efficiency and ease of 

method development in capillary 

electrophoresis (CE) make CE 

attractive for enantioselective

separations. However, CE has 

not been widely used for the

The CE methods developed by CSTL 

researchers provided slightly

different selectivity, thereby reducing 

the likelihood of undetected peak 

overlap. The use of a high sensitivity

UV detection cell dramatically

improved detection capabilities by

increasing peak areas and signal-tonoise ratios. 
determination of enantiomeric purity because the sensitivity 

of the technique is viewed as being inferior to that of liquid

chromatography (LC). In this work, three CE methods were 

developed for the separation of ephedrine and 

pseudoephedrine enantiomers found in plant material, plant

extracts, and dietary supplements containing ephedra. 

Neutral cyclodextrins or mixtures of neutral and charged

cyclodextrins were used as the chiral additive to achieve 

enantioselective separations. Sample preparation involved 

a simple methanol extraction, and a high sensitivity UV 

detection cell allowed detection and quantification of these 

analytes in a variety of complex matrices. The identity of

the enantiomers was confirmed through a standard addition

method. This work has demonstrated that CE can be used 

to determine the enantiomeric composition of chiral analytes 

in complex matrices. The resulting methodology provides

new measurement capabilities that complement existing LC 

and LC/mass spectrometry methods. The analysis of 

ephedra-containing 

materials is important 

from a regulatory standpoint to confirm the natural origin of

the ephedra alkaloids and to determine the levels of 

ephedrine and related alkaloids in dietary supplements. 

35

Use of SRM-Based Column Performance Tests to Guide LC Column Selection in the 

Pharmaceutical Industry. 

L.C. Sander and S.A. Wise (839) 

Chromatographic columns provide the basis for chemical 

measurements in reversed-phase liquid chromatography 

(LC). Columns from different manufacturers or different

manufacturing lots may exhibit a broad range of properties

even though the columns are nominally similar (e.g., C18

columns). Such differences in retention behavior provide 

opportunities for method development and optimization; 

however, variability in LC column manufacture may 

invalidate existing methods and necessitate method

redevelopment. Between-column reproducibility is an

important aspect that must be considered for the

implementation of methods for routine use. Changes that occur in column performance with

usage can also directly affect system suitability. In 2002, the U.S Pharmacopeia 

announced the formation of a Working Group on HPLC Columns. This group was 

charged with “creating categories in order to subclassify the USP designation L1

for C18 (octadecylsilane) HPLC columns.” Several classification schemes are 

currently under consideration in this effort, including approaches based on 

Standard Reference Material column performance tests. 

Analysis of a solid oral dosage 

form of a dietary supplement. 

Peaks are: internal standard (1), 

(-)-ephedrine (2), and (+)-

pseudoephedrine (3). 

10 12 14 16 18 20 22 24 min

1

2

3

Reversed-phase liquid 

chromatography (LC) is a 

mature analytical technique, 

however, analytical methods 

are often developed by trial 

and error, without full 

understanding of the 

fundamental mechanisms that 

make separations possible. 

In FY 03 five Standard 

Reference Materials (SRMs) 

containing ephedra (SRMs 

3240-3244) were certified. 

A clear need exists to distinguish among the hundreds of commercial LC columns that are

available to the analyst. Details of column preparation and other descriptive information are

usually considered proprietary by manufacturers; however, performance based tests provide a

way of characterizing column properties without disclosing such trade secrets. The availability of 

unbiased tests for the assessment of LC column performance are required to facilitate: 1) the 

development and optimization of chromatographic methods by providing a rational basis for 
column selection, 2) evaluation of changes in column performance that occur over the life of the 

column; 3) evaluation of the suitability of columns for specific applications; and 4) characterization

and classification of column properties to permit selection of similar columns among multiple

manufacturers. 

Three Standard Reference Materials have been developed specifically to address the needs of

column characterization. SRM 869a Column Selectivity 

Test Mixture, SRM 870 Column Performance Test 

Mixture, and SRM 877 Chiral Selectivity Test Mixture

offer independent tests to assess column effectiveness 

for the separation of compounds based on differences in

solute shape (“shape selectivity”), general aspects of

column performance (efficiency, retentiveness, activity 

toward bases and metal chelators), and column 

effectiveness for the separation of various classes of

optically active compounds (“chiral selectivity”). The U.

S. Pharmacopeia is currently considering the usage of SRM 869a and SRM 870 to augment their 

L1 designation for C18 columns.

The availability of SRMs to support 

column characterization will greatly

facilitate column selection in 

pharmaceutical and clinical 

applications. Utilization of these 

SRMs by column manufacturers will 

result in the production of more 

reproducible LC columns, which in 

turn will improve the ruggedness of 

methods for routine use. 

Although SRMs 869a, 870, and 877 provide considerable information about column performance, 

other tests may be required to characterize recently introduced columns with new bonding

chemistries. Several classification schemes have been considered, and an approach by Lloyd

Snyder and John Dolan (LC Resources) has considerable merit. This approach uses 10 carefully 

selected solutes to compare column selectivities. An algorithm has been developed to calculate

a single numerical descriptor, which provides an indication of the

relative similarity of two columns. On this basis, decisions can be 

made concerning column choice in method development by 

selection of a column that is similar to one used in an existing 

method, or alternatively, selection of a dissimilar column to one that failed to provide adequate

separation. A large body of data already exists for commercial columns characterized by Snyder 

and Dolan (≈ 150 columns to date). Future work at NIST may be directed towards the 

development of an SRM for LC column classification based on this Snyder/Dolan approach. 

New Glass Filter Reference Materials for Validating the Performance of UV/visible 

Spectrophotometers in the High Absorbance Range 

J.C. Travis, M.V. Smith, and G.W. Kramer (839) 

SRM 2930 is a set of three neutral density glass filters and a blank used to verify the accuracy of

the transmittance/absorbance scales of molecular absorption spectrometers 

(spectrophotometers), and are individually 

certified at five visible wavelengths using the

Reference Spectrophotometer located at NIST.

The 0.1 %, 0.3 % and 92 % transmitting filters of

SRM 2930 bracket and thus extends the range

of transmittance coverage of SRM 930 (10 %, 20

% and 30 % transmitting) and SRM 1930 (1 %, 3

%, and 50 % transmitting). The extension of 

absorbance coverage to three absorbance units 

permits a dynamic range of three orders of 

magnitude in chemical concentration, reducing 

the need to dilute highly absorbing samples for

spectrophotometric analysis and avoiding

needless handling and additional uncertainty. With the 92 % transmitting filter (plain glass) 

The new filters standards will permit 

pharmaceutical and chemical 

producers to validate and quantify a 

product or constituent without an 

intermediate dilution step, subject to 

introducing needless bias and 

uncertainty. The existence of the new 

SRM also adds a traceability link for 

standards producers to provide a 

similar product and leverage NIST’s 

measurement capability. 

36
included in the new set, the total neutral density glass coverage of the three SRMs is now 

consistent with the transmittance calibration range supported by most NMIs who support 

37

The filters will be available for sale in FY 04. 

Production of these filters will not be 

considered continuous, and renewal will be on 

a case-by-case basis. Secondary producers 

will be encouraged to use these filters to 

provide traceability. Recertification services 

will be provided for a few years, after which all 

SRM absorbance filters may be recertified 

through the NIST Calibration service for 

continued traceability support. 

transmittance scales. The nine neutral 

density glass filters of the new set of three

coupled with the older SRMs provide 

coverage of absorbance consistent with

chemical needs and with the measurement 

capabilities of modern instruments. The

software of the NIST Reference

Spectrophotometer (HAS-II) was extended 

to include a stepwise procedure to extend 

the range of qualification of the instrument 

to three absorbance units. The initial 12 

sets of SRM 2930 have been assembled and initial stability, homogeneity, and uncertainty

determination measurements have been made. 

A Fluorescence Assay Designed to Screen for Inhibitors of HIV-1 Nucleocapsid 

Chaperoned Maturation of the Dimerization Initiation Site 

J.P. Marino (831) 

RNA-based drug discovery in the pharmaceutical 

industry requires general approaches for 

detecting and quantifying nucleic acid-protein

interactions that can be used as high-throughput 

screens (HTS). As an extension of our studies of

the dimerization and maturation of the HIV-1 

Dimerization Initiation Site (DIS) Stem-loop, we 

have developed a fluorescence assay to detect

NCp7 catalyzed DIS maturation. Our assay uses 

a molecular beacon approach to detect hairpin

strand exchange associated with NCp7 refolding 

of the DIS kissing dimer. One DIS hairpin is labeled with a fluorescent dye on the 5’-end and the 

static quencher, dabcyl, on the 3’-end. Upon interaction with the second DIS hairpin to form the

kissing dimer complex, the fluorescence emission of the dye 

remains highly quenched and static. In the absence of inhibitor,

NCp7 catalyzed maturation of DIS kissing dimer, that is 

associated with strand exchange between the two hairpin 

stems, results in a burst of fluorescence from the reported dye

as it no longer in close proximity to the quenching agent in the 

mature duplex form (Fig. 1). This fluorescence assay can form 

the basis for a HTS screen to identify novel inhibitors of the 

NCp7 mediated maturation of the DIS dimer. 

The development of specific 

inhibitors of retroviral protein-RNA 

complexes is of significant interest to 

the pharmaceutical industry since 

these complexes provide potential 

novel drug targets. A key roadblock 

to the realization of these goals is the 

availability of rapid and sensitive 

assays to measure and quantify the 

RNA interactions. 

Measurement technology developed in this study will have an 

impact on the biotechnology and

pharmaceutical industries. The 

research is also responsive to

the Advanced Technology 

Program’s interest in projects 

directed at developing novel

approaches for manipulating protein-nucleic acid interactions for possible therapeutic benefits or 

medical diagnostic purposes. 

Example of inhibition of NCp7 catalyzed DIS maturation by

Neomycin at a concentration of 1 μM, as assayed using our 

molecular beacon detection approach. Fluorescence after 

addition of NCp7 to the DIS kissing dimer in the absence

(blue) and presence (red) of Neomycin.
A fluorescence method for rapidly

screening and quantifying HIV-1 NCp7 

chaperoned DIS maturation has been

developed. This method has been 

validated as a screening tool using 

aminoglycoside antibiotics, like 

Neomycin, which have been shown to 

inhibit NCp7 activity

Through collaboration with researchers at the HIV-1 

Drug Resistance Program at the National Institute of 

Cancer, we plan to apply our methods to screen

large public domain libraries of low molecular weight 

compounds. Inhibitors of DIS and/or NCp7 identified

in the HTS screen will then be optimized using

rational design strategies based on NMR structural 

analysis. 

Structure of Pseudomonas aeruginosa PhzD, an Isochorismatase from the Chorismate

Biosynthetic Pathway 

J.E Ladner (831); E. Eisenstein (831/UMBI); J. Parsons (UMBI)

This study provides a description of the enzymes

involved in the chorismate metabolic pathway by 

solving their three-dimensional structures, modeling 

the mechanisms of the chemical transformations, 

and mapping pathway control nodes involved in the 

biocatalytic conversion of glucose to aromatic 

hydrocarbons. Only when the detailed threedimensional structures are known for the enzymes 

can the precise enzymatic mechanisms and 

relationships between the structure and the physical 

properties be predicted. Most recently, the structure

of the product of gene phzD from Pseudomonas aeruginosa has been solved. The phenazines

are biologically active (antibacterial, antifungal, antitumor) aromatic products synthesized mainly 

by Pseudomonas and Streptomyces as part of their chorismate pathways. 

Industrial production of drugs, new 

biopolymers and indigo dyes, can 

be facilitated by increased 

understanding of the aromatic 

amino acid synthesis pathway. The 

increased ability to manipulate this 

pathway through metabolic and 

protein engineering will save nonrenewable, petroleum-based 

feedstock chemicals. 

PhzD isochorismatase

The enzyme on the right (stick model) is from Pseudomonas aeruginosa

part of the pathway that synthesizes a 

bioreactive molecule that has antimicrobial 

properties. The labelled amino acid, 

aspartic acid, initiates the reaction by acting 

like a general acid in the protonation of the 

ether oxygen of the substrate. The 

determination of the crystal structure with 

the substrate provides the basis for 

hypothesizing how the enzyme works and 

thus can provide clues as to how we can 

control or alter its action. 

The work this year has yielded a 

better understanding of the 

chorismate pathway. We have

solved the structure of the product 

of gene phzD and are pursuing both 

biochemical and crystallographic 

methods to determine its role and 

substrate

The synthesis of the aromatic biologically active 

molecules is a very important area of study because 

these pathways do not exist in animals. They exist

solely in bacteria, fungi, and higher plants. The

elucidation of these pathways can provide

pharmaceutical researchers with a better understanding 

for the amplification or inhibition of the production of

these molecules. 

38
Theoretical Protocols for Calculating Structural, Spectroscopic, and Reaction Properties

of Enzyme Active Sites 

M. Krauss (831); Y.S. Lee (NIH) 

39

The reaction path and the electronic structure of enzyme active

sites are analyzed to explore the common electronic character of 

enzyme catalysis. The methodology can be applied to the same

or analogous enzymes within families of organisms or between

different families to discern catalytic motifs and functional

properties of active site residues. The very large size of the

enzyme systems requires a means of dividing them into a

reactive region treated quantum mechanically and the protein 

and solvent environment that is treated classically. Two flavors of

coupling the quantum region to the classical are used in this research. In one, the quantum region 

interacts with effective fragment potentials (EFP) that represent the electrostatic, polarization, and 

repulsive interaction. The EFP are fitted to the quantum interactions for model systems and

produces a consistent interaction across the quantum/classical interface. Classical and 

quantum calculations that determine the qualitatively correct reaction path are

initiated using crystal structures with bound analog inhibitors. The reaction path can

then be used for virtual screening of inhibitors for drug development. The ability to 

calculate the substrate reaction path for homologous enzymes from different types 

of cell organisms and simple mutants provides a flexibility and directness in the 

theoretical virtual screen that should make it very competitive with most experimental programs. 

Applications are to a 

variety of enzyme types 

chosen for their 

importance in validating 

the methodology and 

for applications to 

industrial, military, and 

medical problems. 

Both the QM/MM (Quantum Mechanics/Molecular Mechanics) and EFP 

codes are part of a protocol for studying enzyme behavior. Crystal 

structures with bound analog inhibitors are used to initiate classical and 

quantum calculations that determine the qualitatively correct reaction

path. With the reaction path in hand, optimization and dynamics of the 

entire protein/ligand system with ab initio QM/MM can be considered. 

Also, spectroscopic properties are calculated from both the EFP and 

QM/MM data. 

In bacteriorhodopsin, proton transfer from 

the cyctoplasm to the extracellular side is 

initiated from protonated asp96 in the cytoplasmic region towards the deprotonated Schiff base. 

This occurs in the transition from the late M photocycle state to the N state. A quantum 

mechanics/molecular mechanics (QM/MM) model is constructed from the bacteriorhodopsin 

E204Q mutant crystal structure. Structural transformation occurs in the M state allowing a 

channel to form between the asp96 and Schiff base that permits a water chain between these

groups. Since a part of this channel is lined with hydrophobic residues, there has been a question

on the mechanism of proton transfer in a hydrophobic channel. Using the energy optimized

structures and the molecular dynamics trajectories, the entire vibrational spectrum of

bacteriorhodopsin has been calculated to compare with experimental observations. 

Bacteriorhodopsin (bR) is a naturally occurring 

transmembrane protein that converts light energy 

into metabolic energy. Structural image from PDB. 

Ab initio dynamics using the 

CHARMM/GAMESS 

methodology was used to 

calculate the transfer of the 

proton through a hydrophobic 

channel, a new concept that is 

described in a paper 

submitted to the Journal of the 

American Chemical Society.

Determination of a qualitatively correct reaction path is 

required before a QM/MM calculation can be undertaken. The 

structures of the reaction path in the enzyme active site can

be used to design high-throughput screens for assessing 

reactant, transition state, and product analogues for lead

compounds for drugs. The ability to generally calculate 

vibrational spectra for hydrogen bond networks bound in 

proteins will provide an important tool for analyzing the 

function of these systems. 
The MIRG 2002 Study: Assembly State, Thermodynamic, and Kinetic Analysis of an 

Enzyme/Inhibitor Interaction 

F. Schwarz (831), E. Eisenstein (831,UMBI); D. Myszka and Y. Abdiche (Univ. of Utah); F. 

Arisaka (Tokoyo Institute of Tech.); O. Byron (Univ. of Glasgow); P. Hensley and J. 

Thomson (Pfizer, Inc.; C. Lombardo (The Burnham Institute), W. Stafford (Boston Biom. 

Res. Institute) and M. Doyle (Bristol-Myers Squibb) 

40

Carbonic Anhydrase II

www.ABRF.org 

The pharmaceutical industry has put increased effort 

into the discovery and development of protein-based

therpeutics and reagents (“biologics”) that exhibit

highly specific interactions with their biological targets. 

Fully characterizing the interactions involving 

biomolecules requires information on the assembly

state, affinity, kinetics, and thermodynamics

associated with complex formation. In order to 

evaluate the capabilities and validate the results from 

industrial, academic, and government laboratories to implement analytical ultracentrifugation 

(AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR), the 

Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource

Facilities (ABRF) developed a standardized model system and distributed 

it to a panel of scientists employing these methods. The Association of

Biomolecular Resource Facilities that initiated this study 

is a non-profit standards organization sponsored by 33

companies and consisting of about 800 members from 

industry, academia, and government. 

The analytical technologies in the 

biotechnology industry often 

utilized to measure biomolecular 

interactions include analytical 

ultracentrifugation (AUC), 

isothermal titration calorimetry 

(ITC), and surface plasmon 

resonance (SPR).

The model system consisted of a well-characterized enzyme-inhibitor pair, namely bovine 

carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Accordingly, samples of 

the CBS-CAII model system were distributed to a 

panel of scientists. Study 

participants were asked to 

measure one or more of the 

following: 1) the molecular

mass, homogeneity, and

assembly state of CAII by AUC;

2) the affinity and

thermodynamics for

complex formation by

ITC; and 3) the affinity, 

kinetics, and thermodynamics of complex formation 

by SPR. The results of these measurements were 

evaluated. 

Samples were distributed to a total of 55 

industrial, academic, and governmental 

laboratories that consisted of 7 AUC, 12 

ITC, and 36 SPR laboratories. The 

results of the study showed excellent 

agreement to within 10 % on the binding

affinities and within 2 % on the binding 

enthalpies between the ITC and SPR

laboratories. The AUC results on the 

assembly state of the enzyme were in 

agreement with the ITC results on the 

stiochiometry of the reaction. 

A Metric of Amino Acid Exchangeability Based on Experimental Data 

A. Stoltzfus (831); L.Y. Yampolsky (CARB/UMBI)

The significance of amino acid sequence differences 

between proteins is often interpreted by means of a 

classification of amino acids (e.g., polar vs. non-polar), or

by some continuous measure of amino acid distance.

Available measures based on observed propensities of 

evolutionary change are undesirable because they 

include confounding mutational effects; physicochemical 

Comparative sequence analysis is

a fundamental part of 

computational genomics, 

necessary for genome annotation 

and functional prediction in 

bioinformatics. 
measures are uncertain because they rely on some simplified model of the role of amino acids in

proteins. What is needed is a measure that reliably reflects this role, without the confounding

effects of mutation. 

Methionine is one of 

the essential amino 

acids and must be 

provided by the diet. 

Cysteine is an amino 

acid found in many

proteins throughout 

the body. 

Comparative analysis of protein sequences relies 

on interpreting differences that arise primarily by 

the evolutionary replacement of one amino acid

by another, without the confounding effects of

mutation. How will such changes alter protein 

activity? Probabilities of evolutionary change

reflect, not only effects on the protein, but also

rates of mutation between codons. For example,

the low rate of evolutionary interchange of

Methionine and Cysteine might be due to 

incompatibility at the protein level, but is more 

likely explained by the genetic code, which 

dictates that three separate mutations are

needed to go from a Methionine codon to a 

Cysteine codon. Effective comparative sequence analysis relies on the ability to separate these 

two factors: the exchangeability of amino acids and the mutational exchangeability of codons. 

Prior work on this project resulted in the identification of systematic data on the effects of

switching amino acids in proteins; the development of a statistical method for transforming the

results from different studies to a common scale; the derivation of a measure called the

“experimental exchangeability”, the mean effect EXij of replacing amino acid i with amino acid j, 

based on data from 9671 experimental amino acid exchanges from 12 selected studies; and a 

test for evaluating the power of an exchangeability metric, which 

was used to show that the method for computing EX works.

Also this year, the second phase of the project has begun, with

two demonstration projects to assess the utility of EX in protein 

sequence analysis. The first project addresses the diseasecausing effects of amino acid changes in human genes. For

each type of amino acid change, the observed disease-causing 

propensity is computed from data on known disease mutants 

and SNPs (single-nucleotide polymorphisms). EX outperforms

other possible metrics of severity, accounting for 46 % of the

variance in the logarithm of disease-causing potential. 

The first phase of the project 

was completed this year, 

marked by the submission 

of a manuscript titled 

“Amino acid exchangeability

from experimental data"

Genetics, in review. In 

addition, EX matrix was 

made available to 

researchers in the field of

protein sequence analysis. 

Future plans include more extensive modeling and analysis both of protein mutants and of

evolution, and the modification of methods 

of sequence analysis to take advantage of

an asymmetric measure of pure

exchangeability with statistical weights. 

The evolutionary analysis will determine the 

relative contributions of mutational and 

selective factors in accounting for patterns 

in the evolution of proteins and of proteincoding genes. The analysis of mutants will 

apply simple models for predicting

frequencies of disease-causing alleles, and

will address the long-term independence of

mutational and selective factors, as well as 

relate activity effects to thermodynamic

effects on stability of mutants. 

Amino Acid Exchangeability from Experimental Data Lev Yampolsky and Arlin Stoltzfus

Protein Ref Source species Method 

Sites/

protein Variants

Exchange effects 

assayed 

LacI 2 E.coli sup 328/360 4038 Operon repression, in 

vivo

Lysozyme 3 Phage T4 sup 155/164 1918 Plaque size, in vivo

Interleukin-3 4 H. sapiens sat 103/152 754 Cell proliferation, in vivo

Barnase 5 E.coli sat 109/110 676 RNAse activity, in vivo

β-Lactamase 6

 Pseudomonas sat 27/246 513 Ampicillin resistance, in 

vivo

RecA 7 E.coli sat 20/323 380 Plaque size, in vivo

RTase 8 HIV sat 109/300 366 RTase activity, in vivo

Protease 9 HIV sat 99/99 336 Protease activity, in vivo

Protein V 10 Phage f1 sat 86/87 313 Host inhibition, in vivo

Nuclease 11-13 Staphylococcus scan 143/149 290 ∆∆G, in vitro

HGH 14 H. sapiens scan 50/191 50 Dissociation constant, in 

vitro

Insulin 15 H. sapiens scan 37/51 37 Receptor affinity, in vitro

Total 9671

EX EXS BLOSUM PAM250 -lnMJup 1/GRA

1.6E- 75 7.6E- 47 2.2E- 49 3.4E- 37 3.9E- 21 1.8E- 06

C S T P A G N D E Q H R K M I L V F Y W EXs

C . 0.28 0.12 0.20 0.35 0.28 0.11 0.11 0.29 0.38 0.26 0.29 0.25 0.15 0.11 0.35 0.06 0.35 0.35 0.18 0.28

S 0.35 . 0.48 0.20 0.49 0.40 0.38 0.37 0.30 0.31 0.31 0.33 0.27 0.37 0.27 0.25 0.35 0.29 0.26 0.16 0.32

T 0.30 0.40 . 0.15 0.40 0.33 0.23 0.22 0.19 0.26 0.22 0.27 0.22 0.15 0.23 0.25 0.37 0.24 0.23 0.07 0.27

P 0.35 0.38 0.28 . 0.44 0.39 0.35 0.29 0.36 0.38 0.36 0.26 0.22 0.29 0.23 0.26 0.41 0.31 0.27 0.35 0.33

A 0.37 0.37 0.33 0.23 . 0.39 0.41 0.27 0.24 0.28 0.28 0.26 0.20 0.61 0.25 0.30 0.35 0.28 0.26 0.25 0.29

G 0.27 0.30 0.26 0.14 0.37 . 0.28 0.21 0.21 0.28 0.24 0.19 0.23 0.27 0.14 0.19 0.21 0.17 0.18 0.24 0.24

N 0.25 0.35 0.29 0.25 0.40 0.39 . 0.20 0.25 0.29 0.24 0.25 0.18 0.27 0.19 0.23 0.23 0.21 0.24 0.12 0.27

D 0.26 0.26 0.20 0.20 0.31 0.29 0.19 . 0.33 0.26 0.28 0.23 0.19 0.16 0.26 0.22 0.18 0.21 0.22 0.09 0.25

E 0.38 0.38 0.29 0.19 0.53 0.42 0.26 0.50 . 0.37 0.40 0.34 0.35 0.21 0.36 0.37 0.34 0.38 0.39 0.12 0.38

Q 0.38 0.42 0.36 0.21 0.50 0.42 0.34 0.07 0.42 . 0.40 0.39 0.34 0.50 0.47 0.40 0.60 0.35 0.35 0.16 0.38

H 0.26 0.35 0.20 0.18 0.47 0.37 0.22 0.14 0.25 0.31 . 0.24 0.26 0.31 0.20 0.36 0.23 0.28 0.23 0.08 0.28

R 0.20 0.24 0.20 0.13 0.43 0.23 0.07 0.12 0.22 0.28 0.24 . 0.31 0.07 0.12 0.24 0.20 0.21 0.25 0.06 0.24

K 0.32 0.34 0.40 0.24 0.58 0.52 0.36 0.35 0.25 0.38 0.34 0.43 . 0.33 0.43 0.30 0.42 0.33 0.33 0.17 0.38

M 0.36 0.33 0.44 0.08 0.37 0.22 0.54 0.39 0.27 0.37 0.27 0.13 0.13 . 0.55 0.52 0.58 0.36 0.29 0.63 0.31

I 0.35 0.19 0.23 0.14 0.32 0.16 0.17 0.05 0.19 0.18 0.21 0.12 0.11 0.28 . 0.42 0.50 0.33 0.31 0.07 0.25

L 0.37 0.22 0.20 0.15 0.34 0.20 0.20 0.11 0.20 0.25 0.28 0.19 0.17 0.36 0.32 . 0.30 0.33 0.28 0.18 0.25

V 0.34 0.28 0.48 0.17 0.36 0.23 0.10 0.35 0.15 0.24 0.22 0.17 0.16 0.57 0.54 0.29 . 0.21 0.19 0.31 0.25

F 0.20 0.18 0.51 0.13 0.27 0.09 0.19 0.06 0.06 0.18 0.24 0.12 0.09 0.37 0.30 0.32 0.38 . 0.32 0.44 0.22

Y 0.15 0.17 . 0.15 0.36 0.29 0.13 0.09 0.18 0.29 0.19 0.26 0.17 0.39 . 0.30 . 0.34 . 0.30 0.23

W 0.15 0.09 0.02 0.07 0.06 0.19 . . 0.07 0.06 0.23 0.10 0.05 0.11 . 0.22 0.11 0.38 0.28 . 0.15

EXd 0.31 0.30 0.30 0.18 0.41 0.32 0.25 0.24 0.24 0.29 0.28 0.25 0.21 0.31 0.30 0.30 0.32 0.28 0.26 0.20 0.28

EX, Experimental Exchangeability

Data on Experimental Exchanges 

Evaluation: 1. Predict results of target study with EX* computed from other studies

2. Compare predictive power of EX* to other available predictors. 

0

0.1

0.2

0.3

0.4

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5

EX

All

Buried

Exposed

Change in Volume, Å3

Statistical

test P value

Target study

EX*

Best predictor

41
Biological Macromolecule Crystallization Database 

G.L. Gilliland, M. Tung, T.N. Bhat, and J.E. Ladner (831) 

The NIST/CARB Biological Macromolecule Crystallization 

Data (BMCD) contains crystal data and crystallization 

conditions for biological macromolecules. The BMCD 

includes the crystallization data for peptides, proteins, 

protein-protein complexes, nucleic acids, nucleic acidnucleic acid complexes, protein-nucleic acid complexes, and 

viruses for which diffraction quality crystals have been 

obtained and reported in the literature. Besides collecting

data from the literature, data can be acquired from other

resources such as the Protein Data Bank (PDB). The

mapping between BMCD data model and mmCIF

(macromolecular Crystallographic Information File) representation of data of PDB entries resulted

in a new and expanded data representation for the BMCD. This new representation of data gives

a consistent way to carry out data exchange between the BMCD and other structural biology 

resources, and hence provides a new avenue for BMCD data acquisition. This new 

representation of data is not only compatible with the earlier BMCD data conventions, but also 

with the PDB data. The data model simplifies and reduced the number of manyto-many relationships making the database much easier for data management. 

For example, tables for depositors and auditing information have been added.

With this new data model, data to be added to the BMCD can be extracted from 

the PDB mmCIF data set using a Star (CIF) Perl API. 

The BMCD was designed as 

a tool to assist x-ray 

crystallographers in the 

development of protocols 

to crystallize biological

macromolecules, those that 

have previously been 

crystallized, and those that 

have not.

42

This new data model for the BMCD has resulted in

improved data management capabilities. In addition,

data to be added to the BMCD can be extracted from the

PDB mmCIF data set using a Star (CIF) Perl API. Since a 

significant fraction (15 % to 20 %) of the PDB contains

comprehensive crystallization information, the BMCD can be 

significantly expanded using the new model. 

http://www.nist.gov/srd/biotech.htm 

The PDB resource contains 

more than 18,000 crystals for 

which data can be extracted. 

Even tapping into the wealth of information in the PDB, much of the crystallization data required

to reproduce the experiments will still be lacking from the majority of entries. The missing

information includes the crystallization method, crystal growth temperature and pH, and chemical

additives. Rather than collecting this information from the literature, which is very time consuming, 

a data mining approach using data clustering will be

carried out. Data clustering will be based on the

characterization of crystals and molecules, using such

data items as unit cell parameters and macromolecular or

protein properties. A new BMCD website will be created 

that will deliver the results of clustering rather than just the

individual entry information, and will also allow more 

complex queries. 

Future Plans: A new generation 

data model called a “Data Mart”

will be employed with the BMCD 

website to provide new ways of 

analyzing and querying the 

underlying data. 

Predicting Vibrational Spectra of Dissolved Salts 

L. Wang (831); K.K. Irikura (838) 

Details provided in the Exploratory Research Section
Structure to Function: Using a Structural Genomics Approach 

G.L. Gilliland and J.E. Ladner (831); A. Teplyakov, G. Obmolova, O. Herzberg, J. Orban, 

and J. Moult (UMBI); A.J. Howard (Illinois Institute of Technology). 

With large-scale genomic sequencing efforts meeting

with success, post-genomics research efforts are

focusing on functional assignment of gene products. One

approach is the use of structural data determined by 

crystallographic or NMR methods. In the work described

here, CSTL researchers and collaborators have 

elucidated or are in the process of determining more

than forty protein structures. 

43

Schematic representation of

the Haemophilus influenzae

KW20 Rd genome. 

The folding pattern of the threedomain is shown as a ribbon 

representation of the polypeptide 

backbone of the polymer. The 

secondary structure elements, αhelices and β-strands, are indicated. 

Many of the gene products of completely sequenced

organisms are "hypothetical", that is they cannot be 

related to any previously characterized proteins, and so are of unknown structure and function. In

an effort to address this issue, an interactive website, entitled “Structure 2 Function” was

developed collaboratively by NIST, UMBI, and the Industrial 

Macromolecular Crystallography Association. Structure 2

Function (S2F) is a structural 

genomics project that aims to solve

structures of these poorly characterized proteins by using X-ray 

crystallography and protein NMR techniques. More than 30 

scientists participate in the compilation of this structural data,

and are experts in NMR, protein purification, gene cloning,

crystallization, bioinformatics and proteomics. The project 

status is updated daily. 

Research involving the largescale structure determination 

of hypothetical proteins from 

Haemopilus influenzae or 

homologs from other 

organisms has been underway 

to assess how structural 

information can influence 

functional assignment. 

http://s2f.carb.nist.gov/ 

The initial set of targets was selected from the first completely 

sequenced bacterial genome of the Haemophilus influenzae. 

Structures of hypothetical proteins are being used to guide

further studies and narrow the field of such studies for

ultimately determining protein function. 

As an example, a specific protein (YchF) from Haemophilus 

influenzae was cloned, expressed, and the crystal structure 

determined. The polypeptide chain is folded into three domains

having a crab like structure. The crab-like three-domain structure 

suggests where the binding site for double-stranded nucleic acid

is located. Fluorescence measurements have demonstrated the

ability of this protein to bind a nucleic acid duplex, and have also

shown the specific binding of GTP (guanosine 5’-triphosphate). 

Taken together with other experimental data and given the 

universal phyletic distribution of this family of proteins, these 

results suggest that YchF may be part of a ribonucleoprotein 

complex and may function as a GTP-dependent 

translation factor. 

This project involving a structural genomics approach 

has demonstrated that working on a number of proteins

in parallel, chosen on the basis of a unifying scientific 

theme, is a powerful strategy. 
Future Plans: The approach developed in 

this project will focus next on alternative 

splicing, a new and important scientific 

discovery, resulting from the analysis of the 

genomic scale sequence information on 

higher eukaryotes. 

A large body of structural information has been 

obtained for hypothetical bacterial proteins while 

methods development to accomplish large-scale

structure determination are on going. In addition

to aiding in functional assignment, this effort is 

identifying new protein folds and a number of

possible new targets for drug development. 

Cluster SIMS Depth Profiling of a Polymer Drug Delivery System 

C.M. Mahoney, S. Roberson, and G.J. Gillen (837) 

Secondary Ion Mass Spectrometry (SIMS) has 

already proven to be a powerful tool in the

spatial analysis of drug delivery materials.

However, with the advent of cluster primary ion

beams, such as SF5

+

, the ability to determine 

the distribution of a drug as a function of depth

becomes feasible for the first time. With SIMS, 

the distribution of both drugs and excipients 

within a drug delivery system can be

determined with a high degree of spatial resolution (<1μm) and sensitivity (as low as ppm) when 

compared to other analytical methods (such as Raman or IR). 

CSTL researchers developed a cluster 

SIMS technique to aid pharmaceutical 

companies in the development of new 

drug delivery systems, including complex

controlled release formulations, which 

are highly dependent upon the 

homogeneity and diffusivity of the 

materials involved.

The behavior of a series of poly(lactic) acid (PLA) films doped with varying concentrations of the 

model drug 4-acetamidophenol, under SF5

+

 primary ion bombardment, was investigated in order 

to determine whether the distribution of the drug could be measured as a function of depth. The

figure shows a typical depth profile of a PLA film containing 20 % by weight 4-acetamidophenol (≈

200 nm) spun cast onto a Si wafer, and shows that the secondary ion intensities associated with 

both polymer and drug moieties remain stable with increasing SF5

+

 primary ion dose. These

secondary ion intensities remain constant until reaching the PLA / Si interface, at which point both

PLA and 4-acetamidophenol intensities drop,

corresponding to an increase in Si secondary 

ion intensity. The polymer film / Si interface

was surprisingly well defined (≈ 10 nm to 15 

nm depth resolution) which may imply that

sputter-induced topography formation was 

not a significant limitation. Several other 

biodegradable materials including

polyglycolic acid, polylactic glycolic acid,

polycaprolactone and polyethylene glycol 

were also depth profiled, with similar results 

to PLA. These results suggest that the

common main chain structural unit of these 

polymers, R-CO-O-R, facilitates beaminduced bond cleavage and damage removal. 

44

Future plans involve utilizing the signal

enhancement obtained with cluster primary 

ion beams for improvements in secondary ion

imaging of drug delivery systems. Once this 

is achieved, we will explore the possibility for 

3D molecular characterization of these 

systems. The utilization of SIMS for quantification of drugs in complex drug delivery matrices is

also an important part of our future research plans. Collaborations with key pharmaceutical 

SIMS depth profile of a PLA film containing 20 

% by weight 4-acetamidophenol (≈ 200 nm) 

spun cast onto a Si wafer. The film was 

analyzed using SF5

+

 primary ion bombardment.
These results indicate that in these 

particular model systems, the 

distribution of the drug as a 

function of depth can be monitored 

when applying cluster SIMS. 

manufacturers will be initiated so that we can utilize 

the cluster SIMS capability for applied problem 

solving and to help with the characterization and

development of new drug delivery materials. 

Impact Energy Dependence of SF5

+

 Ion Beam Damage of PMMA Films Studied using ToFSIMS

M.S. Wagner (837) 

Recent advances in instrumentation for Secondary Ion Mass Spectrometry (SIMS) have focused

on the application of polyatomic primary ion sources for enhancing the molecular secondary ion 

signals of organic materials. A few studies have also noted that 

some polymers, in particular poly(methylmethacrylate) (PMMA),

display the unusual characteristic of maintaining their molecular

ion signals after extended bombardment with polyatomic ion

beams. In this study, the damage of ≈ 70 nm thick spin-cast films 

of PMMA by SF5

+

 with impact energies from 2.5 – 8.75 keV was 

studied using Time-of-Flight (ToF) SIMS. Sputtering of the 

polymer using SF5

+

 was alternated with collection of static positive and negative ion ToF-SIMS 

spectra to assess the damage created by SF5

+ ion bombardment in the ion dose range of 2.5 x 

1013 to 5 x 1014 SF5

+

 ions/cm2. All beam energies displayed maintenance of the characteristic 

molecular ion signals for PMMA despite extended SF5

+

 bombardment. An increase in the SF5

+

bombardment energy increased the sputter rate of the polymer without increasing the 

accumulation of beam-induced damage. Additionally, neither fluorocarbon cations nor anions 

were observed in the ToF-SIMS spectra of the damaged PMMA films. 

CSTL researchers 

studied the effect of 

SF5

+

 impact energy in 

the range of 2.5 keV to 

8.75 keV on the damage 

of PMMA thin films. 

The figure below shows that as the SF5

+

 primary ion dose increased, the C4H5O+ secondary ion 

intensity could be described by three regions. Region (i) is characterized by an initial intensity 

decrease followed by region (ii) that shows a quasi-stabilization of the secondary ion intensity.

After this extended stable regime, region (iii) is characterized by a rapid decay in C4H5O+

secondary ion intensity as the Si+

 secondary ion intensity increased, indicating that the polymer 

film had been sputtered through and the silicon substrate had been exposed. However, the slope

b) a) 

The (a) C4H5O+ (m/z = 69) and (b) Si+ (m/z = 28) secondary ion intensity (normalized to its initial 

intensity for each damage profile) as a function of SF5

+

 ion dose for impact energies between 2.5 

keV and 8.75 keV. The SF5

+

 impact energy is marked on each of the damage profiles. The C4H5O+

secondary ion is characteristic for the PMMA film while the Si+

 secondary ion is characteristic 

for the silicon substrate. The three regions of the damage profiles are marked in (a) for the 5 keV 

SF5

+

 damage profile. 

45
46

of region (ii) in the damage profiles did not increase as a function of SF5

+

 impact energy, 

suggesting that changing the impact energy did not change the rate of damage accumulation in 

the polymer film. These results suggest that the highest impact energy available would be optimal 

for molecular depth profiling of polymer films. 

The measurements reported here have demonstrated that the degree of damage accumulated by 

the PMMA film was not affected by the SF5

+

 impact energy. However, increasing impact energy 

increased the sputter rate. These results have provided optimized conditions for depth profiling of 

polymer samples, with the anticipation of aiding in the development of polymeric biomaterials and 

drug delivery vehicles.
4. Chemical and Allied Products 

47

The need for NIST–characterized 

zeolite reference materials was the 

consensus of an NSF-sponsored 

meeting that included representatives 

from academia and industry. 

Dark field scanning transmission electron 

microscopy image of faujasite zeolite.

Often referred to as the keystone 

industry because nearly every

sector of the manufacturing 

economy uses its products, the 

U.S. chemical industry ranks 

among the world’s leading 

industries, accounting for an 

estimated one-quarter of total 

world chemical production. 

CSTL provides reference data,

theoretical models, and artifact

standards that are important to

the chemical industry. These

standards are used for process

design and quality control, and

help ensure traceability of field

measurements to reliable, worldrecognized standards. 

Zeolite Reference Materials: Chemical Properties, Structural Parameters, and Particle Size 

Distribution 

S. Turner, R.A. Fletcher, E.S. Windsor, and R.R. Cavanagh (837); J.R. Sieber, T.W. Vetter, 

R.L. Zeisler, and D.A. Becker (839); B.H. Toby and J. Kelly (MSEL); S.D. Leigh (ITL); M.

Davis (Caltech); S. Yang and A. Navrotsky (Univ. California at Davis) 

Zeolites encompass a large class of hydrated

silicate materials that have structures containing

channels and cages of various dimensions. Both 

synthetic and natural zeolites have widespread

industrial applications in petroleum refining, 

chemical manufacturing, gas separation and 

dehydration, environmental cleanup, and as

ingredients in detergents. Despite their widespread use over the past three to four decades, no

materials with characterized physical parameters have been available for intercomparison

studies. 

Three zeolites were studied: faujasite, Linde Type A, and

ZSM-5. To deal with their hydroscopic nature and its

effect on chemical and structural characterization,

materials were handled in a humidity-controlled

environment with minimal exposure to ambient 

environmental conditions. Major elements were 

determined by x-ray fluorescence analysis, gravimetry,

and instrumental neutron activation analysis (INAA), 

while trace element concentrations were determined by 

INAA. Structural parameters were characterized using

synchroton x-ray diffraction and neutron diffraction. 

Particle size distributions were characterized by

optical and sedimentation techniques. 

Reference and information values related to the chemistry, structure and particle size distributions

have been obtained. The materials will provide a basis for an intercomparison of zeolite 

measurements between analytical laboratories. They will also be available to customers as NIST

Standard Reference Materials. 
Characterization of Spatial Variations in Nomex® Fiber Crystallinity with Confocal Raman 

Microscopy

C.A. Michaels, D.B. Klinedinst, E.S. Windsor (837); and D.B. Chase (DuPont Co.)

48

Confocal Raman microscopy has been used to study 

spatial variations in crystallinity for spun Nomex® 

polymer fibers provided by collaborators at DuPont.

The spinning process is thought to yield a "skin" region 

on the outside of the fiber with crystallinity differing 

from that of the bulk. Because of its impact on the

material performance properties there is significant

interest in probing the skin properties as a function of processing parameters. The subtle 

dependence of the Raman spectra of this material on 

crystallinity is exploited to allow imaging of crystallinity 

changes across polished fiber cross sections. Images 

acquired with 1 μm2 lateral resolution reveal no 

detectable skin, thus setting an upper limit on its size

for the fibers studied. Ongoing efforts will focus on

higher resolution imaging of these materials and those 

prepared under varying processing conditions. These

aramid fibers are widely used in materials for their fire

retardant properties, in applications ranging from 

protective apparel for firefighters to composite aircraft

parts. The degree of crystallinity of these fibers has a 

significant effect on aspects of material performance

including mechanical strength, chemical resistance

and dyeability, and can be modified through variations in processing conditions. It has been 

shown that changes in the fiber crystallinity result in subtle changes in the Raman spectra, thus

providing a non-destructive means for measuring this 

property with spatial resolution of approximately 1

μm. Raman imaging of the fiber skin requires a 

sample preparation method that allows measurement 

of the fiber cross section. Several approaches have 

been used, including polishing of epoxy mounted

fibers and microtome sectioning of epoxy mounted

fibers. Analysis shows that the epoxy matrix yields 

no significant Raman signal and thus does not

interfere with the measurement. As configured, the

microscope probes a lateral sample area of 1 μm2

with a sampling depth approximately 5 μm. Careful 

analysis of the variation of aramid band intensities 

across this fiber cross-section yields no evidence for

the existence of the postulated skin. This 

measurement does not preclude the presence of a

skin smaller than the probe volume; however it does

set an upper limit on its size for this type of fiber. 

Measurements aimed at refining this limit are 

ongoing and are also being extended to fibers 

prepared under different processing condition. 

CSTL scientists used Confocal 

Raman microscopy to image the 

variation in crystallinity for 

DuPont Nomex® fibers used for 

protective clothing. 

NOMEX® polymer fibers 

Raman Shift (cm-1) 

Normalized Raman spectra of high (blue) 

and low (red) crystalline content Nomex® 

fiber acquired with 785 nm excitation. 

32 x 32 μm Raman image 

of a polished Nomex®

fiber cross section

mounted in an epoxy

matrix plotted on a color 

scale ranging from black 

(minimum intensity) to 

red (maximum intensity)
Advanced Experimental Capabilities for Fluid Properties Measurements 

R.A. Perkins, A. Laesecke, J. Magee, M. McLinden, S. Outcalt, and J. Scott (838); S. Altintas 

(PREP Postdoc); A. Abdulagatov (Russian Academy of Sciences); C. Lösch (Rubotherm) 

49

Advances in measurement 

technology enable new and 

improved measurements of 

the thermophysical properties 

of gases, liquids, and 

supercritical fluids.

The need for measurement standards as well as data

needs of the chemical, energy, refrigeration, and 

aerospace industries continues to drive the development of 

new measurement devices for the thermophysical 

properties of fluids. Unique apparatus are under 

development in CSTL for improved measurements of

density, sound speed, critical points, and phase 

equilibrium. In addition to high accuracy, the goals in this effort are for rapid measurement, a

small sample size for rare and/or hazardous substances, and materials for construction that will 

withstand corrosive fluids. Density. A two-sinker densimeter (photo at left) demonstrated a 

reproducibility of 0.0002 kg m-3, representing <1 part per million 

for liquids and about 20 parts per million for gases at 1 MPa. 

Critical Points. Under development is a short-residence-time 

flow apparatus to rapidly measure the critical temperature up to 

1000 K and pressure up to 10 MPa for small fluid samples with 

an uncertainty of 0.08 K and 0.01 MPa. 

Sound Speed. A cylindrical sound speed apparatus, based on

the pulse-echo technique, has been developed for liquid 

measurements from 90 K to 310 K. Tests on liquid propane 

verified that the uncertainty in sound speed is less than 0.05 %.

Phase Equilibria. A new apparatus was demonstrated using 

propane to measure the phase equilibrium where multiple liquid 

phases coexist with the vapor phase. This apparatus operates 

from 200 K to 400 K and utilizes a moveable capillary line to 

sample each phase. 

An apparatus developed along these lines has been used to

establish several de-facto standards for the thermophysical 

properties of fluids. These standards allow the calibration and

performance verification of devices used to measure the

thermophysical properties of fluids. Data measured with these

apparatus along with evaluated literature data are used in NIST

databases such as REFPROP, which provides convenient access 

to the most accurate property data available. 

Sinkers and magnetic suspension coupling at the heart of the densimeter. The electromagnet at

the top hangs from a balance and levitates the permanent magnet below it. The permanent

magnet and sinkers are normally contained within a pressure vessel. The two sinkers, a balance,

the magnetic suspension coupling, and a mechanism to pick up each sinker constitute the

density measuring system. 

International Standards for Refrigerant Properties 

M.O. McLinden and E.W. Lemmon (838)

Knowledge of the thermophysical properties of any fluid in a thermodynamic cycle is required to

evaluate the energy efficiency, capacity, etc. Standards exist for determining and reporting the 

performance of air-conditioning systems at standard ratings conditions. However, differences 

among multiple property formulations lead to different performance ratings. This is especially 

problematic in international trade involving refrigerants and was the prime motivation for ISO to

develop a standard for refrigerant properties. 
The new ISO Standard, entitled “Refrigerant

Properties,” has progressed to the status of a 

Draft International Standard in just over three 

years. The ISO Working Group first had to decide 

on the scope of the Standard (including which

properties and which fluids to include). The group

then had to determine how to define the 

properties (e.g. in terms of experimental data, published equations of state or other models, or

some sanctioned computer package). NIST staff were the lead authors of the Standard as well as 

key technical experts in the Working Group (WG) discussions. The widespread use of the NIST

REFPROP database was acknowledged in the WG discussions. 

A new ISO standard for the 

thermodynamic properties of 14 key 

refrigerants (10 pure fluids and four 

refrigerant blends) has been 

completed and is currently in review 

by the international community. 

The properties for the refrigerants R12,

R22, R32, R123, R125, R134a, R143a,

R152a, R717 (ammonia), and R744 

(carbon dioxide) and the refrigerant blends 

R404A, R407C, R410A, and R507A were

felt to be sufficiently well known to be

included. Older refrigerants, such as the

CFC R12, were included for comparison

with the newer HFCs, such as R134a. All of the key thermodynamic properties, in single-phase 

states and along the saturation boundary, are included. The

Standard defines the properties with tables and by defining

the equations used to generate those tables. The equations 

of state and mixture model are from the published literature,

and, for completeness, all numerical coefficients are also

given. 

This new Standard, if accepted, will define the 

properties used by the air-conditioning and 

refrigeration industry for the performance rating

of their equipment. It will remove the longstanding potential for contention and confusion 

arising from different manufacturers using 

different property formulations. 

Although the Standard does not specify any particular computer package, the models specified in 

the Standard are the same ones used in REFPROP. Thus, REFPROP will be compliant with the

new standard; this will elevate the current status of REFPROP as a de facto standard to one with

a true international sanction. This is a fitting acknowledgment of the 15 years of effort, which 

NIST has devoted to the properties of the “alternative” refrigerants. 

The Industrial Fluid Property Simulation Challenge 

R. Mountain, A. Chaka, R. Johnson, D. Friend, J. Magee, L. Watts, T. Bruno, R. Chirico, A. 

Laesecke, E. Lemmon, and M. McLinden (838); J. Olson, J. Moore, and D. Frurip (Dow 

Chemical); M. Schiller (Dupont); J. Golab (BP); F. Case (Colgate-Palmolive); R. Ross (3M); 

P. Kolar (Mitsubishi Chemical) 

50

The demand for accurate physical property 

data far exceeds our capacity to supply it

experimentally. This is especially true for 

unusual, novel, or toxic materials and mixtures, 

or state points far from ambient where it can be 

challenging or expensive to obtain experimental

data in a timely manner.

Accurate physical property data in chemical 

product and process design are often 

inaccessible from experiments. Important

data include the solubility of oxygen in fluids 

at elevated temperatures or pressures and 

the viscosity and thermal conductivity of 

fluids at elevated pressures. 

Molecular simulations hold great promise to meet this growing need for property data. Methods 

based on molecular dynamics and Monte Carlo algorithms in particular have substantial potential 

for predicting thermal and transport properties of dense fluids. It is not clear, however, that this

approach is suitably mature for industrial applications. The First Industrial Fluid Properties
Simulation Challenge was organized by NIST and collaborators from industry (Dow Chemical, 

DuPont, Colgate-Palmolive, and BP) to assess the capabilities of various simulation techniques. 

The challenge was sponsored by a committee of the American Institute of Chemical Engineers to

predict certain liquid properties for specified conditions. 

51

The Industrial Fluid Properties 

Simulation Challenges are organized 

by NIST and several companies (Dow 

Chemical, DuPont, Colgate-Palmolive, 

BP, and now 3M and Mitsubishi 

Chemical) to assess the current 

capability of molecular simulations to 

make useful predictions. 

The First Challenge revealed that existing 

simulation methods for predicting the shear

viscosity may be too inefficient, prompting an

interest in improving sampling methods. Detailed

reports on the first Challenge will be published in

a special issue of Fluid Phase Equilibria. The 

Second Challenge was opened in September 

2003 and will run until September 2004. The

challenge has generated interest and support in 

academia and industrial R&D for improving force 

laws and for improving the efficiency of simulation sampling methods. 

Topics for the Second Challenge: 

• Vapor pressure and heat of 

vaporization for acetone and 

butyramide (shown in figure) at 

elevated temperatures 

• Henry’s Law constants for gases in 

ethanol at elevated temperatures 

• Heat of mixing for butylamine with 

heptane 

http://www.cstl.nist.gov/FluidSimulationChallenge 

Transport Property Models for Refrigerants

M.L. Huber, A. Laesecke, and R. Perkins (838) 

Transport properties, such as viscosity and thermal 

conductivity, are important in the design of 

chemical processing equipment for various 

industrial sectors including the petroleum refining, 

transportation, energy, and refrigeration. The NIST 

computer program REFPROP (NIST REFerence

fluid PROPerties) has become the global de-facto

database standard for modeling refrigerant

properties. We have developed new models and 

correlations that improve the representation of the

viscosity and thermal conductivity surfaces for 

refrigerant fluids and have coded them into REFPROP. 

A combined correlational/predictive 

model was used to predict transport 

properties for the many fluids of 

industrial importance that lack wideranging property data. We then 

coded the models in REFPROP, the 

NIST computer program and 

database for thermodymamic and 

transport properties of fluids. 

The method of extended corresponding states (ECS) was the modeling approach that proved 

successful in providing viscosity and thermal conductivities for fluids that lack wide-ranging and 

accurate data. This modeling method applies a technique in which one obtains estimates of the

properties of a fluid of interest by mapping onto the properties of a well-characterized fluid known

as the “reference fluid”. We developed a high-accuracy correlation for the viscosity surface of 

R134a (figure at right with viscosity along z-axis), an important fluid used in many applications 

including automobile air conditioning units, supermarket display cases, and in health care, as a

propellant for asthma inhalers. R134a is also important as a reference fluid for ECS models to
predict properties of similar, but less 

well-characterized halocarbon fluids. 

The new surface represents the 

viscosity to within 2 % along the liquid 

saturation boundary and 0.5 % in the 

dilute vapor region. (The blue/red line 

in the figure indicates the transition 

from vapor (blue) to liquid (red) at the

critical point.) We also developed a

high-accuracy correlation for the 

viscosity surface of R125, another 

important refrigerant fluid that is a 

component of refrigerant blends such 

as R407C and R410A that may be 

used as replacements for R22. We

then applied the ECS model to 17 

additional pure halocarbon fluids, and 

developed improved surfaces for 

calculating the viscosity and the 

thermal conductivity of these fluids.

High-Throughput Liquid Transport Properties Measurements in Membranes and Films 

C. Muzny, S. Outcalt, J. Scott, and L. Watts (838); R. Hedden (854) 

52

Measurements of diffusion and sorption for 

specific liquids and gases in thin layer films are 

important for characterizing membranes and films

according to their transport properties. These 

measurements provide a means to delineate

design criteria for mass separating agents such as

membranes and polymeric adsorbents by 

compiling the effects on transport behavior from 

both chemical and structural subgroups in the

material. 

Although polymeric and inorganic 

materials are used in membrane and 

adsorptive separation processes, the 

lack of predictive capabilities for the 

transport properties of mixtures in 

any selected material is a significant 

barrier to the optimum use of existing 

materials and development of new 

materials. 

A major product of this project is an automated highthroughput apparatus for measuring the diffusivity and

solubility of a variety of test components in a wide 

variety of membranes. The technique employs sixteen

test cells that are temperature controlled individually 

and that can be loaded with different solvents, test

solutions, and membranes. The figure below shows 

the robotic system for the apparatus.

A critical component of this method is to have easily 

detectable, standard test compounds that probe 

different physical and chemical characteristics of the 

membranes. The testing of this instrument using

standard test fluorophores has continued. The 

molecular diffusivity of G4 PAMAM dendrimers that

are used as test fluorophores was characterized via 
fluorescence correlation spectroscopy. This novel technique provides the fundamental 

measurement needed for membrane transport characterization of a molecular species. With the 

instrument operating in a continuous test and data

production mode, numerous operational problems

were identified. One issue, fluorescence stability, 

was largely addressed by testing a range of buffer 

solutions in order to control the pH. Automation 

issues have also been solved, and the instrument 

has now been operating stably for periods of up to 

one month. Final launch of the web-based database for membrane transport data produced by 

this instrument is the next major goal. 

The availability of data and predictive

methods from this effort will impact 

industries that use membrane-based 

separations including water purification, 

pharmaceuticals, and chemical 

manufacturing. 

Measurements, Modeling, and Data for Pressure-Driven Membrane Separations 

C. Muzny, H. Sun, and J. Moon (838); J. Cho (Univ. of Colorado); M. Chapman (U.S. Bureau

of Reclamation); and J. Ranville (Colorado School of Mines) 

53

The ultimate goal of this work is to 

provide a standard test mixture for 

testing membrane fouling along with 

standard procedures for evaluating 

membrane performance. 

The accepted physical property used industrially to 

characterize membranes is the pore size

distribution or molecular weight cutoff. Since the

concept of pore size distribution has no industrial 

standard, we have developed a set of standard

membranes with highly regular pore structures for 

use in the development of standard membrane characterization methods. These are inorganic 

membranes with pores in the 10 nm to 100 nm size range and with very narrow pore diameter 

distributions. 

The standard test solutions and 

procedures resulting from this work will 

provide industry with a uniform method 

for pressure-driven membrane 

separation performance testing. 

The liquid-liquid porosimetry apparatus for physical 

characterization of membranes that we previously

developed has been used as the principle

characterization method for these standard

membranes, although field-emission scanning

electron microscopy has also been used for pore 

size verification. Membrane testing has also included a fouling study using uniformly sized

polystyrene beads as a model foulant. By looking at both monodisperse and polydisperse foulant

solutions, we have been able to test the validity of a standard fouling model on particles in the

10 nm to 100 nm size range. Characterization of these fouling solutions has also included field

flow fractionation, and 

correlations between the results 

of the field flow fractionation and

flux reduction continue to be 

studied. As a final test of the

overall goal of providing 

standards for pressure driven 

membrane performance testing,

a small-scale membrane module 

test bed is now in service and 

being used to test the 

performance of spiral wound,

reverse osmosis modules under 

the standard conditions.

The flow field flow fractionation technique is being used as 

an idealized system for the modeling of fouling during 

pressure driven membrane separations. 
Surface Oxygen Chemistry of the Gas-Sensing Material SnO2 (101) 

A.M. Chaka (838); U. Diebold, and M. Batzill (Tulane University) 

Stannic oxide (SnO2) is a widely used material for 

gas-sensing devices that are based on metal-oxides. 

As an oxidation catalyst, SnO2 is sensitive to the 

adsorption of oxidizing and reducing gases. A 

change in the electrical resistance of polycrystalline 

SnO2 films occurs if the composition of the ambient 

gas changes. Improving the sensitivity and chemical 

differentiability of metal oxide sensors has been difficult due to the complexity of the

polycrystalline films and poor understanding of the 

mechanisms responsible for performance. 

Understanding the fundamental 

mechanisms of gas sensors at the 

atomic level can lead to the design 

of more sensitive and robust 

sensors with greater chemical 

differentiation.

It is not known which crystalline faces of polycrystalling SnO2

are responsible for sensor activity, nor what the mechanisms 

are. During the past year experimental observations a SnO2

single crystal using various microscopic and spectroscopic

techniques (e.g., scanning tunneling microscopy and low 

energy loss diffraction) have identified the (101) surface as 

one which undergoes significant changes at elevated 

temperatures in the presence of low O2 pressure. The 

crystalline surface changes dramatically yet reversibly when 

the O2 partial pressure is varied around 10-12 Pa while heating 

from 550 K to 680 K. 

SnO2 single crystal

First principles modeling identified a facile oxidation/reduction reaction as oxygen occupied or 

vacated bridging sites on the surface. The stoichiometric Sn4+O2 surface is stable only at high

oxygen chemical potential. As pO2 is decreased below a threshold pressure, a Sn2+O bulk

termination becomes the most favored thermodynamically (figure below). These two surfaces 

interconvert without reconstruction by occupying and vacating bridging oxygen sites. The

calculated differences in free energy between the two surfaces predict a temperature threshold of 

680 K at which the interconversion occurs, which is consistent with experimental results. This 

variability of the surface composition is possible because of the dual valency of Sn (Sn2+ or Sn4+) 

and is essential for the application of this material in gas-sensing devices. 

Future plans call for calculating the

band structure and density of

states at the material’s surface.

These calculations will link the 

differences in surface stoichiometry and oxidation state with

observed changes in conductivity 

as a function of oxygen partial 

pressure and temperature. 

Additional crystal faces will be

investigated ((110), (100) and 

(001)), as well as adsorption of CO 

and NO for sensor application.

54
The NIST WebBook* and the IUPAC/NIST Chemical Identifier - Standards and Delivery 

Mechanisms for NIST Chemical Reference Data 

P. Linstrom, D. Tchekhovskoi, S. Stein, and G. Mallard (838)

The NIST Chemistry WebBook remains one of 

the most used resources for chemical and 

physical property data. Usage has continued to

grow at about 10 % to 20 % per year, currently 

over 650,000 distinct IP addresses access the 

WebBook per year. We continue to see a very

high rate of return users indicating a level of

satisfaction with the WebBook. Usage continues to grow for all of the components

of the WebBook. 

Committees of IUPAC (Steve Stein, 

Chairman) and ASTM (Peter Linstrom, 

pivotal member) have pursued INChI 

and the web-based standards for 

chemical information communication.

55

IUPAC

A major part of this project is to find and

evaluate thermochemical and thermophysical

property data - with a major emphasis in the

evaluation process being the deployment of automated tools 

for that evaluation. The second part is to make this and 

other data such as infrared (IR), ultraviolet (UV) and mass 

spectra available and easy to access. The identification and

communication of this information has required the 

development, testing and implementation of algorithms for 

extracting a unique chemical identifier from the chemical

structure. 

Finding the enormous amount of 

thermochemical data (heats of

formation, entropies, heat 

capacities, heats of reaction) as 

well as thermophysical property

data (vapor pressure, viscosity, 

boiling points, melting points, 

etc) that are available to the 

technical community is time 

consuming and expensive. 

More effort has gone into acquiring data and a major donation of infrared spectral data from the

Dow Chemical Company was received this year. In addition, the work on the IUPAC/NIST

Chemical Identifier (INChI) has continued. CSTL scientists continue to work on the INChI along

with data-exchange standards for chemical data, which is an essential part of making the 

WebBook a leading part of the next generation of electronic chemical information. 

During 2003 the eighth edition of the NIST Chemistry WebBook* was released. In addition to an

enhanced profile of prediction tools, a major increase in the infrared spectra was made with the 

release of the digitized Coblentz Society infrared spectral data. This data had only been available 

in printed form and the release of the data in fully digitized electronic form represents a major 

addition to the resources of the WebBook. Perhaps as a result of the

increase in IR data, the IR usage increased more than the overall usage. 

The finalization of the donation of over 50,000 IR spectra by The Dow 

Chemical Company to NIST culminated a multiyear effort. Historically the

large collections of data that are present in the chemical company records have not been

available to the public. The decision by Dow to donate the data and the decision to make it

available through NIST and the WebBook represent a major vote of confidence for the WebBook. 

*http://webbook.nist.gov/chemistry

Predicting Vibrational Spectra of Dissolved Salts 

L. Wang (831) and K. K. Irikura (838) 

Details provided in the Exploratory Research Section.
Thermophysical Properties and a Data Retrieval System for Ionic Liquids 

J. W. Magee, M. Frenkel, R. D. Chirico, Q. Dong, A. Laesecke, M. O. McLinden, R. A.

Perkins, J. A. Widegren, D. G. Archer (838); and G. J. Kabo (Belarusian State U., Belarus) 

Ionic liquids are a class of organic salts that

are liquid at or near room temperature. 

They are generally composed of a large 

asymmetric organic cation and either

organic or inorganic anion. As such, they 

are non-volatile and non-flammable.

Therefore, they have been proposed as 

solvents for Green Processing. The 

physical properties of ionic liquids may be

tuned, for example by altering the hydrophobicity, and this affects solubility, reaction rate, and

selectivity. In spite of the advantages, fundamental data on their physical and chemical properties

are scarce and a knowledge base of

standardized systems is needed that would 

lead to a comprehensive database for U.S. 

industry to exploit these solvents. 

Ionic liquids have been proposed as 

solvents for Green Processing. To provide 

U.S. industry with the knowledge base to 

exploit these solvents, we have begun to 

study the physical properties of ionic liquids, 

and to develop both standardized systems 

and a comprehensive data retrieval system. 

56

To achieve these goals and reach an

international consensus, two IUPAC projects 

have been initiated with CSTL involvement. The 

first project, Thermodynamics of Ionic Liquids, Ionic Liquid Mixtures, and the Development of

Standardized Systems (2002-005-1-100), addresses the concern that currently there are no 

internationally adopted standards for reporting thermodynamic data for ionic liquids. To address 

this issue, a standardization mechanism for reporting thermodynamic data for ionic liquids could

be accomplished by an expansion of the Guided Data Capture software developed by the NIST

Thermodynamics Research Center. These issues were discussed by members of the project’s 

task group, which included CSTL staff, at a workshop of the 17th IUPAC Conference on Chemical 

Thermodynamics (Rostock, Germany). Outcomes of this workshop were reported in a recent 

issue of the Journal of Chemical and Engineering Data. The second IUPAC project, Ionic Liquids 

Database (2003-020-2-100), presents a vision for a distributed-access data retrieval system for 

ionic liquids and their mixtures that encompasses chemical structures, solvent properties, ionic 

liquids in synthesis, reviews, reactions and catalysis, manufacturer information, benchmark

properties and models, and

thermophysical and thermochemical

data. 

CSTL has played a key role in recently

approved projects under the auspices of the 

International Union of Pure and Applied 

Chemistry (IUPAC) that will provide 

recommendations of standardized systems 

for data and will develop a comprehensive 

database for ionic liquids. 

In addition to the IUPAC projects, NIST

continues to provide benchmark 

physical properties measurements for 

selected ionic liquids. For the archetypal 

ionic liquid, 1-butyl-3-methylimidazolium 

hexafluorophosphate, we published the 

first reports of thermodynamic properties 

for the ideal state at temperatures to 

1500 K and high-accuracy

thermodynamic properties in condensed

states (crystal, glass and liquid) 

covering a range of temperatures from 5 

to 550 K.

Publications

• Preface to Special Section: Papers Presented at the 

Workshop on Ionic Liquids, ICCT, Rostock, 

Germany, July 28 to August 2, 2002. J. W. Magee,

J. Chem. Eng. Data 2003, 48, 445. 

• Thermodynamic Properties of 1-Butyl-3-

methylimidazolium Hexafluorophosphate in the 

Ideal Gas State. Y.U. Paulechka, G.J. Kabo, A.V. 

Blokhin, O.A Vydrov, J.W. Magee, and M. Frenkel, 

J. Chem. Eng. Data 2003, 48, 457-462.

• Thermodynamic Properties of 1-Butyl-3-

methylimidazolium Hexafluorophosphate in the 

Condensed State, G. J. Kabo, A. V. Blohkin, Y. U. 

Paulechka, A. G. Kabo, M. P. Shymanovich, J. W. 

Magee, J. Chem. Eng. Data 2004, in press.
Evaluated Molecular Structures and Vibrational Frequencies for C1-C2 Chlorocarbons 

D.R. Burgess, Jr. and J.A. Manion (838) 

57

This work is a compilation of 

gas-phase molecular geometry 

and vibrational frequency data 

for all industrially important C1

and C2 chlorocarbons, 

including radicals. 

Chlorinated hydrocarbons are used throughout the

chemical industry, both as end products and as base 

chemicals in the production of refrigerants, plastics,

pesticides, solvents, and other products. Reliable

molecular property data are needed to derive accurate 

thermodynamic information for use in models of industrial 

production, to better understand the fates of chlorinated

compounds in the environment, and to improve waste destruction methodologies. 

• Models for predicting the thermal 

decomposition of chlorinated hydrocarbons 

in waste destruction are very dependent on 

accurate thermochemical functions for 

stable and metastable chlorinated 

hydrocarbon species. 

• Thermochemical properties such as the 

equilibrium constant for formation strongly

depend upon specific molecular properties 

such as the molecular vibrational 

frequencies.

We recently reviewed the experimental

information on the enthalpies of formation of 

the stable C1 and C2 closed-shell chlorinated 

hydrocarbons. Presently, molecular geometry 

and vibrational frequency data for C1 and C2

chlorocarbons have been compiled and

evaluated. The available experimental data

were supplemented with ab initio and density

functional theory calculations both from the 

literature and from our own work. Comparisons 

between experimental and calculated data

were made to verify the experimental data, to

validate the calculated values, and to provide theoretical values in the absence of experimental 

data. We have attempted to correct the theoretical geometrical parameters and vibrational 

frequencies for systematic differences by benchmarking the calculations using similar molecules 

with reliable experimental data. Based on the evaluated experimental data, supplemented with

those from calculations, recommended values for the molecular structure and vibrational 

frequencies (including torsion and inversion modes) are given for each of the molecules. 

The C1 chlorocarbons include the chlorine-substituted methanes,

methyl radicals, methylenes, and the 

chloromethylidyne radical. The C2

chlorocarbons include the chlorinesubstituted ethanes (figure left), ethyl

radicals (figure right), ethenes, 

ethenyl radicals, ethynes, and the 

chloroethynyl radical. We also include 

data for the unsubstituted, reference 

compounds (e.g., methane, methyl 

radical, ethane, ethyl radical). 

Thermodynamic Interpretation of Low-Pressure Ion-Molecule Reactions 

K K. Irikura (838)

Thermochemical data are used to determine 

the feasibility and the thermal requirements 

of chemical reactions. Part of the quality 

assurance for thermodynamic data 

compilations such as the NIST WebBook 

involves understanding the proper 

procedures for analyzing experimental data. 

Gas-phase studies of reactions between 

ions and molecules have been a rich source

of thermochemical data for more than 30

years. Indeed, they are a major part of the 

NIST Chemistry WebBook. Surprisingly, 

there are two competing procedures for 

extracting thermodynamic data from the

experimental observations. However, only 
one of these procedures can be correct. In practice, the two procedures usually give similar 

results, so the fundamental confusion in interpretation has been neglected. However, for some 

types of reaction (those with large entropy changes) the two methods of analysis give widely 

different results. 

The following two reactions in the gas phase 

present problems in interpretation of the 

thermochemistry:

HBr+

 + CF4 → CF3

+

 + HF + Br (1) 

CH5

+

 + SF6 → SF5

+

 + HF + CH4 (2)

The procedures for extracting thermochemical 

data are based on an interpretation of enthalpy 

change or free energy change. In both

reactions above, the “enthalpy” interpretation

infers that DH is less than or equal to zero,

while the “free energy” interpretation infers that 

DH is less than or equal to zero. From the 

Gibbs equation, results in the thermochemistry 

differ by TDS, which is large when DS is large.

The figure below shows the inferred ionization

energy (y-axis) of CF3 from Eq. 1. The red

experimental points (before) were obtained

using the "enthalpy" interpretation. The points 

themselves are limiting values from 

experiments, whereby other values would be in

the direction of the arrows and, hence, tend to

be smaller. If they are later interpreted by the 

"free energy" method (after), the red limiting data points move upward (corresponding black 

limiting points) and become consistent with other experimental and theoretical results. 

This work (J. Am. Chem. Soc., 121, 7689-7695, 1999) has stimulated at least one additional 

experimental study and an opposing opinion. To reply, in preparation is an additional publication 

with additional evidence and arguments in favor of one interpretation. 

Evaluation of Free Radical Standard Potentials 

P. Neta and R. E. Huie (838)

58

Standard electrode potentials for 

a broad set of organic and 

inorganic free radicals are being 

compiled and evaluated as part 

of an international collaboration 

under the auspices of IUPAC. 

The standard electrode potential of a substance is a

fundamental thermodynamic quantity that represents 

the tendency for a molecule to gain or lose electrons. It

is widely used for predicting the course of chemical 

reactions. Although standard electrode potentials have

been extensively evaluated and tabulated for stable

species, the situation is much less satisfactory for 

reactive intermediates such as free radicals. The

explosive growth in biochemical research exploring one-electron oxidations and reductions 

highlights the need for further evaluation of standard potentials for free radicals. Toward this 

effort, an IUPAC Task Group that included CSTL representatives was formed with the objectives 

of updating the 1989 standard electrode potential compilations and developing a consistent set of

recommended potentials for a wide range of organic and inorganic radicals in aqueous solutions.

The first major phase of this activity, data compilation and evaluation for the major 

organic and inorganic radicals, has been largely completed. Standard potentials 

for a number of organic radicals derived from phenols, anilines, indols, and related

antioxidants were evaluated based on various equilibrium studies and

recommendations presented to the Task Group. As well, standard potentials were 

derived for the solvated electron and the hydrogen atom, which are particularly 

important since they form an anchor point for the reducing end of the electrode

potential scale. Other members of the IUPAC Task Group outside of CSTL have dealt with a

 IUPAC 
59

The isodesmic computational 

approach used here is based on 

combining accurate experimental 

knowledge of reactivity in one or 

several reactions of the same class 

with computational information 

based on quantum chemistry. 

range of other organic and inorganic free radicals, including the important hydroxyl radical, which

anchors the oxidizing end of the electrode potential scale. The planned CSTL contribution to this 

IUPAC effort includes an extension to

aromatic organochalcogens and

organoradicals derived by oxidation 

and the inorganic P, As, Sb, Bi 

radicals. We plan also to work with the 

Task Group to optimize the standard 

potential scale and establish a NIST 

web site for the dissemination of these

results.

Isodesmic Reactions for Transition States (IRTS) 

V.D. Knyazev (838) 

The isodesmic approach to studying

thermochemistry relies on the conservation of bonds 

in reactions, with the same number of each type of

bond (single, double, triple) occurring in both

products and reactants. The technique of isodesmic

reactions for transition states (IRTS) has been 

developed and validated for calculation of reaction

barriers and rate constants. This computational approach is based on combining accurate

experimental knowledge of reactivity in one or several reactions of the same class with

computational information based on quantum chemistry. The IRTS technique has been shown to

yield the energy barriers and rate constants for abstraction reactions of two classes (H + 

chloroalkanes and Cl + halomethanes) with a high degree of accuracy. For example, average 

onstants for seven H + chloroalkane

reactions considered are 17 % to 24

%, depending on the quantum

chemical method used. In contrast, a 

non-isodesmic quantum mechanical 

approach typically results in a severalfold difference between calculated and

experimental rate constants. Accuracy 

in reaction barrier calculations at the 

level of a few kJ mol

deviations between calculated and experimental rate c

he results will have an impact on the 

chemistry, nanotechnology, and biochemistry. We also intend to develop an IRTS tool for

exploring reactivities in going from small to large molecular systems. 

-1 has been

demonstrated for the Cl +

halomethanes reaction. 

T

field of chemical kinetics by providing 

a predictive computational tool for a

wide range of chemical reactive

systems such as those used in 

materials chemistry, nanotechnology, 

polymer chemistry, biotechnology, and 

soot/pollutant formation. We plan to

extend the field of application of the 

IRTS technique to reactions involving

large molecules relevant to polymer 

This activity is expected to have an immediate impact 

in the increasingly important field of physiological free 

radical chemistry. It is also expected to have an 

impact on the development of advanced oxidation

technologies, which are typically based on free radical 

chemistry, and antioxidants for use in stabilizing 

organic systems such as polymers. 

The figure shows calculations of the rate constant for 

several hydrogen abstraction reactions as compared 

with experimental data (symbols). Different line styles 

correspond to different levels of quantum chemical

theory used. J. Phys. Chem. A, 106, 11603-11615 (2002). 
60
5. Energy and Environmental Technologies 

Many CSTL activities impact the energy industry sector,

including reference data and models, fuel SRMs, metering

standards, and flow calibrations. NIST’s accurate and reliable

flow standards and databases are critical to this industry both for 

process optimization and for use in custody transfer applications.

CSTL also provides standards that allow this industry to verify 

compliance with environmental regulations. The Environmental 

Technologies industry sector, part of an estimated global market 

of $530 billion, depends on NIST/CSTL for world recognized

artifact standards as well as reference data and models to

demonstrate compliance with environmental regulations, and for reliable decision-making for 

environmental remediation, waste disposal, emissions trading and other applications.

Natural Gas Flow: North American Laboratory Comparison Project 

P. I. Espina (836) and W. F. Guthrie (ITL) 

The natural gas industry requested that NIST

develop a round robin testing program to

ensure the quality of measurements in the 

three major natural gas flow calibration

facilities in North America: namely, the 

Colorado Engineering Experiment Station, 

Inc. (CEESI), Ventura, IA, TransCanada

Calibrations Ltd. (TCC), Winnipeg, Manitoba,

Canada, and Southwest Research Institute 

(SwRI) Metering Research Facility, San Antonio, TX. 

In 2004, 750 billion cubic meters of natural 

gas will be consumed in the USA (24 % of 

our energy needs). Consumption is 

projected to increase by 50 % by the year 

2025. Metrology is necessary to assure 

proper custody transfer of the resource in 

the North American natural gas industry. 

With the support of Daniel Industries (a flow meter manufacturer in 

Houston, TX), NIST developed the transfer standard package used in 

this laboratory comparison (schematic shown), one of the largest 

transfer standards ever used in a NIST calibration program. The

transfer standard was composed of a turbine meter separated from a

multi-path ultrasonic flow meter by a flow conditioner. The transfer 

standard had a diameter of 300 mm, a length of 9.7 m, and a mass of

4,000 kg. 

TCC

The transfer standard package, which has volumetric flow range from

726 m3

 h−1 to 6,169 m3 h−1, was instrumented with a NIST-developed 

sensor package that included pressure, temperature, and frequency 

instrumentation controlled by a laptop computer. 

After agreement among the stakeholders, the transfer standard was 

extensively tested prior to the start of the comparison to assess and

quantify its repeatability, with hysteresis effects, its day-to-day, flowno flow-flow, and mount-dismount-mount reproducibilities, and its 

sensitivity to flow profile effects. With this knowledge, the transfer 

standard was used to compare the performance of the laboratories by 

testing on 5 occasions: SwRI (Oct. 2002) – CEESI (Nov. 2002) –

CEESI (May 2003) – SwRI (May 2003) – TCC (Aug. 2003). In

addition, the package was tested for pressure effects at SwRI, which 

was the only laboratory capable of varying line pressure. The results 

Sensor Package 

61
will be used to (i) quantify the level of comparability among calibration laboratories to improve 

future performance, (ii) guide future laboratory comparison projects, and (iii) guide the upcoming 

CIPM Key Comparison on natural gas flow being led by PTB/Germany and NMi/Netherlands. Flow Conditioner

T T

3.75"D 6.25"

Ultrasonic Flow Meter

3 D

Turbine Meter

3 D 5 D 5 D 6.25"

CSTL researchers provide reference 

correlations for solubility of important 

gases in water at high temperatures. 

These standards will find use in 

numerous areas of high-temperature 

aqueous physical chemistry, 

particularly in the steam power industry 

and in geochemical applications. 

High-Temperature Reference Correlations for the Solubility of Gases in Water 

A.H. Harvey (838); R. Fernández-Prini (CNEA, Argentina); J.L. Alvarez (CNEA)

In the steam power industry, it is important to

know the distribution of solutes such as O2 and 

CO2 between liquid water and steam at high

temperatures. The solubility of gases in water is 

an important parameter in the design of many

industrial and environmental processes. These

quantities are also important in geochemical 

modeling, particularly for common geologic 

gases such as CH4, CO2, and H2S. For all of

these applications, it is useful to have reliable, 

evaluated data in the form of a correlation covering the entire temperature range of interest, 

which extends to near the critical temperature of water. 

CSTL researchers took advantage of two relatively

recent advances to produce state-of-the-art correlations. 

First, our collaborators in Argentina have developed

improved methods for reducing experimental data to the

thermodynamic quantities of interest (the Henry’s

constant and the vapor-liquid distribution constant).

Second, previous work at NIST has produced theoretical 

constraints on the high-temperature behavior of these

properties as the solvent critical point is approached. 

This leads to improved functional forms that better

describe the high-temperature data and have superior

extrapolation behavior for systems where the range of high-temperature data is limited. 

We conducted a thorough literature search and 

found usable high-temperature data for 14

solutes in ordinary water and 7 solutes in heavy 

water. We evaluated the data, converted them 

to the appropriate thermodynamic quantities, 

and produced correlations for the Henry’s

constant and vapor-liquid distribution constant, with both correlations based on the same final 

data set. The correlations developed in this work have been incorporated into an official guideline

that will be adopted by the International Association for the Properties of Water and Steam at its 

2004 meeting. 

New reference correlations for the Henry’s 

constant and the vapor-liquid distribution 

constant have been published: 

Fernández-Prini, Alvarez and Harvey, J. 

Phys. Chem. Ref. Data 32, 903-916 (2003). 

5 D 6.25"

P

5 D 6.25"

P

Schematic of Transfer Standard (flow from left to right)

62
Influence of Oxygen on the Shelf Life of Coal SRMs

R. Zeisler (839) and W.D. James (Texas A&M) 

In 1997 users of SRM® 1632b (Trace Elements in Coal), 

issued in 1985, reported a change in the measured 

values of the carbon mass fraction with the new values

being about 1.6 % lower than previously measured. 

Additional evidence of change was provided by trace 

element data in this material, which indicated

approximately the same change in mass fraction values 

for a number of elements determined by INAA, although

the new measurements were still within the bounds of

uncertainty given in the certificate. While a change in carbon content through loss of CO2 may 

have been a plausible explanation for lower than previously measured carbon values, the 

measured decrease of a larger number of elemental content values may be explained by a gain 

of matrix mass, such as by irreversible oxidation and therefore increased matrix mass. To directly 

measure mass fractions of oxygen in coal samples, a fast (14-MeV) neutron activation analysis 

(NAA) procedure was employed. The procedure was applied to test samples of SRM 1632c

Trace Elements in Coal (Bituminous).

Twenty-six samples each were 

selected and compared for three

storage conditions: (1) in liquid nitrogen

vapor and (2) at room temperature in 

the original containers packaged under

argon, and (3) at 50 °C and open to air 

to accelerate aging. The 14-MeV NAA 

is an effective procedure for the direct 

determination of oxygen. It

demonstrated sufficient precision and 

accuracy to determine a relative 

change of about 5 % or more, in the 

oxygen mass fraction of about 11 %, 

which is equivalent to about 0.5 % of

the total mass. To evaluate the sample 

stability, test points were set at 6 and 

54 months for the oxygen

determination, more frequent 

gravimetric tests were carried out for 

the samples in the open 50 °C storage.

The gain in mass and in oxygen for the latter samples is illustrated in the figure above. All gain in 

mass can be explained by the gain in oxygen content. No change in oxygen content was

measured in the other storage conditions. The measurements give assurance that properly 

packaged coal SRMs, protected from oxygen and heat, are stable during their shelf life. 

-1

0

1

2

3

4

0 10 20 30 40 50 6

Change in Mass of SRM 1632c with Time

in Accelerated Aging (50 °C) Storage

Ž mass (%)

Month

Mass after prescribed drying is set to "0"

0

Change in mass

calculated from measured

change in oxygen fraction

Gravimetry

CSTL scientist and collaborators 

validated the long-term stability 

of a coal SRMs under proper 

storage. Samples were found 

stable during the more than fouryear observation, except for the 

ones stored open at higher 

temperature. 

Determination of Hg in Crude Oils in the Department of Energy Strategic Petroleum 

Reserve by ID-CV-ICPMS 

W.R. Kelly, S.E. Long, and J.L. Mann (839) 

CSTL researchers have developed a 

highly accurate technique for 

measuring mercury in crude oil. Prior 

to this work, Hg in this matrix has 

been a major analytical challenge. 

The Clean Air Act as amended in 1990 required

the U.S. Environmental Protection Agency (EPA) 

to submit a report to Congress on Hg emissions 

to the atmosphere. The EPA responded with the 

Mercury Study Report to Congress that identified

63
a number of sources of Hg emissions to the atmosphere

and quantified those emissions with the best available

estimates. However, no estimates were given for crude 

oil consumption because of the lack of reliable data. It is

well established that coal is a major source of Hg 

emissions to the atmosphere. The Hg content of coal 

combusted in the US is about 100 ng g-1. The EPA,

concerned with the well-known toxic behavior of Hg, now requires reporting of Hg emissions from 

companies that process more than 10 pounds of Hg per year, 

and this reporting requirement may be a prelude to regulation of 

Hg emissions. If the mean content of Hg in crude is 10 ng g-1, 

as commonly believed, then all refineries would be over the limit. 

Given the fact that the magnitude of

crude oil consumed in the US is 

equal to that of coal, many

scientists have speculated that 

crude oil could be a major unknown

component to the global Hg cycle.

Isotope dilution cold vapor inductively coupled plasma mass 

spectrometry (ID-CV-ICPMS), a highly accurate analytical 

method developed at NIST was used in this study. The high

accuracy of this technique is superior to existing techniques 

because of three unique capabilities: 1) very low and

controllable blanks which average 3 ± 2 pg (± 1s), 2) 

high sensitivity of the ICPMS, and 3) a digestion method

using only high purity nitric acid in closed system (Carius tube) which prevents loss of the highly 

volatile Hg and prevents contamination of the sample. 

DOE’s Strategic Petroleum Reserve

The Hg concentration determined in 

almost 100 different crude oils from the

U.S. Strategic Petroleum Reserve (SPR)

by ID-CV-ICPMS, populate a new 

database and allow for more strategic 

decision-making. The Hg concentration

ranged from 0.02 ng g-1 to 10 ng g-1

(figure) with about 80 % of the samples 

falling below 2 ng g-1, much lower than

previously reported. 

These measurements constitute the 

first and only high-accuracy database

on Hg in crude oils indicates that the

Hg concentration is about an order of 

magnitude lower than currently

believed.

Absorption Coefficient Measurements of Aerosol Particle Agglomerates 

C. Presser (836); J. Conny (837); A. Nazarian (SAIC) 

Details provided in the Exploratory Research section 

64
A Question of Accuracy: How Does Thermal-Optical Analysis for Climatically Important 

Black Carbon in Atmospheric Soot Behave Optically? 

J. M. Conny (837) 

CSTL researchers test critical assumptions

regarding the optical behavior of Thermaloptical analysis (TOA), which impact its 

accuracy. This is critical since TOA is a 

principal method in the U.S. for measuring 

refractory carbon in atmospheric soot. 

Refractory black carbon (BC) in atmospheric 

particulate matter presents a formidable 

measurement challenge for climate change 

researchers. Optical methods for determining 

particulate carbon mass require prior 

knowledge of the absorption and scattering

cross sections, the latter which varies widely 

with the chemical composition of particles. Thermal-optical analysis (TOA) measures lightabsorbing refractory carbon mass but does not require prior knowledge of the optical cross 

sections. However, TOA incorporates critical assumptions about the optical behavior of organic 

carbon (OC) and BC. This work tests these assumptions and pursues an approach to optimizing 

TOA for accuracy through an assessment of how the absorptivity of particulate carbon behaves 

during analysis. 

In TOA’s light transmission method (TOT),

refractory BC is distinguished from OC through a

protocol of thermal desorption accompanied by 

pyrolysis in helium and thermal oxidation in a HeO2 flow. As carbon evolves from a filter sample it 

is detected by flame ionization detection. Pyrolysis of OC generates char, which is indicated by 

the attenuation of 670 nm laser light. BC is differentiated from OC when the attenuated laser 

signal returns to the level prior to the initiation of the heating protocol, indicating that an amount of

carbon equivalent to the original OC component has been removed from the filter sample and

only the carbon mass equivalent to BC remains. 

TOA has an essential optical component; 

therefore, it plays an important role in 

climate change studies for determining the 

mass of light-absorbing aerosol carbon. 

Accuracy in TOT is hindered by the 

positive bias from unpyrolyzed OC 

measured as BC and by the negative

bias from the unaccountable oxidation 

of BC at high temperatures during

analysis. TOT assumes that the 

absorptivity of the pyrolized OC 

remains constant at high temperatures 

and that BC and pyrolized OC have

the same absorptivity. In this work, the 

attenuation due to particulate carbon 

absorption was isolated from the total 

attenuation, which included scattering 

by non-carbonaceous material. Particulate carbon absorptivity was then calculated by dividing the

laser attenuation due to carbon by the carbon mass which gives the apparent specific absorption

cross section (σC). The figure shows how σC typically varies during the course of analysis. 

Assessment of σC at critical points in TOT provides a robust approach for adjusting TOT’s 

temperature protocol (blue area in figure) to insure consistency in TOT’s optical behavior and to 

achieve accuracy in atmospheric BC measurement. 

65
Structure and Reactivity of the Hydrated Hematite (0001) Surface 

A.M. Chaka (838); T. Trainor, P. Eng, and M. Newville (Univ. Chicago); G. Waychunas 

(Lawrence Berkeley National Lab); J. Catalano and G.E. Brown (Stanford Univ.) 

An understanding of metal oxide 

structure and chemistry in a realistic 

environment at the nanoscale will help 

in the development of robust predictive 

models for creating catalysts, coatings, 

sensors, microelectronic components, 

and corrosion inhibitors, as well as in 

efforts to remediate polluted soils, 

ground water, and surface water. 

Reactions at metal-oxide surfaces are of great

significance in the chemistry of contaminants in 

aquifers and soils, as well as 

industrial/technological processes involving

catalysis, microelectronics, and passivation of 

metals. Hematite (α-Fe2O3) and the isostructural 

corundum (α-Al2O3) are among the most

common metal oxides in industry and in soils, 

but exhibit drastically different chemistries for 

reasons that are not understood. 

Knowing how the structure and reactivity of a 

metal oxide surface change upon exposure to 

water enables better design and control of 

surface properties for industrial conditions.

The types of surface functional groups exposed at metal-oxide/water interfaces are likely to differ 

substantially from models based on ideal surface terminations of the bulk crystal and should

result in dramatic differences in reactivity compared with clean or dehydroxylated surfaces. The

combination of theoretical and experimental work performed in a wet environment has provided

the first evidence for the fundamental mechanisms involved in the early stages of hydrolysis of

iron atoms in iron oxide. Using crystal 

truncation rod diffraction (CTR) and ab initio 

thermodynamics (first principles density 

functional theory (DFT) electronic structure

calculations linked to finite temperature and 

pressure thermodynamics), it was determined that the stable hydrated hematite (0001) surface 

consists of two domains – one with Fe-rich regions terminated by singly coordinated hydroxyls, 

and one with oxygen-rich regions terminated by doubly and triply coordinated hydroxyls. The

presence of singly coordinated hydroxyls on hydrated α-Fe2O3(0001) and their absence on

hydrated α-Al2O3(0001) explain the higher reactivity of the former. DFT calculations indicate that

water reacts with hematite both heterolytically and

homolytically at a threshold water pressure orders of 

magnitude lower on α-Fe2O3(0001) than on αAl2O3(0001). The electronic structure of corundum 

does not support the homolytic dissociation

mechanism of water. 

Phase diagrams of the surface free energies 

calculated as a function of water vapor pressure and 

in equilibrium with liquid water (see figure), as well 

as surface relaxations, were used to identify the

structure of the two domains observed in the CTR 

experiment on the hydrated hematite surface and to 

account for the difference in reactivity between Fe

and Al oxide. 

Hematite (0001) Surface Energy Phase

Diagram in Equilibrium with O2 and H2O 

66
Chemical Kinetic Database for PAH formation for Heptane Combustion 

V. Babushok and W. Tsang (838) 

A new approach has been developed that extends 

the possible use of molecular simulations to include 

polycyclic aromatic hydrocarbon (PAH)/Soot

formation processes. In combination with the

capability of existing databases to cover oxidations, 

this represents at least in principle the complete 

picture for the combustion with heptane as a fuel. 

CSTL researchers build a kinetic 

database for heptane combustion 

that can be used to simulate both 

the oxidative properties (ignition 

delay, flame velocities) as well as 

the PAH/Soot forming propensities 

of the system. 

In studying the combustion of heptane, particular

emphasis was placed on the beta C-C bond scission reaction and isomerization of the radical

intermediates in the decomposition. These include, the four heptyl and 2 pentyl radicals as well 

as all the 1-olefinyl radicals, 1-butenyl-3, 1-butenyl-4, 1-pentenyl-3, 1-pentenyl-4 1-pentenyl-5, 1-

hexenyl-3, 1-hexenyl-4, 1-hexenyl-5, and 1-hexenyl-6. 

The effects of energy transfer leading to pressure dependencies have also been considered.

These reactions lead to the small, unsaturated compounds and radicals that are the inputs to

existing PAH/Soot models. The model was validated against existing data on ignition delays (from 

OH) and flame velocities. Some problems on existing databases for the latter have been

identified and modifications have been made to fit results under rich conditions. 

Our approach is being used by engineers at Wright-Patterson

Air Force Base to model the impact of the use of nitromethane

additives on soot formation. Future plans are two-fold. First, a 

large extension of the present work is planned to cover an 

assortment of the components of a real fuel mixture that can

serve as a surrogate, which was a focus of discussion at the 

workshop “Combustion Simulation Databases for Real 

Transportation Fuels“ sponsored by NIST in FY03. Second, 

the optimization and extension of existing databases are planned so that current data and

concepts are included.

Existing mechanisms for the 

combustion of heptane have

been extended to include the 

reactions involved in the 

formation of the precursors of 

PAH/Soot. Cracking reactions 

of heptane were also involved.

OH Kinetics – Evaluation of Data and Experimental Techniques 

V.L. Orkin and M.J. Kurylo (838) 

CSTL researchers critically reviewed the 

determination of lifetimes for chemicals 

emitted into the atmosphere has been, with 

particular reference to measurement 

methods and techniques for hydroxyl 

radical kinetics and procedures for 

estimating the ozone depletion potential 

and global warming potential. 

Reactions of the hydroxyl radical in the gas 

phase are central to atmospheric chemistry 

and combustion processes. We have been 

engaged for a number of years in carrying 

out accurate and precise kinetic 

determinations, and also in developing

computational procedures for estimating 

these values. One of the major reasons for 

carrying out these studies is the important 

role of OH in establishing the atmospheric 

lifetime of chemical compounds, which is the key parameter in estimating environmental impact

due to their emission to the atmosphere, ozone depletion potentials (ODPs) and global warming 

potentials (GWPs). 

67
M.J. Kurylo and V.L. Orkin "Determination of Atmospheric 

Lifetimes via the Measurement of OH Radical Kinetics," 

Chem. Rev., 103, 5049-5076 (2003). 

Over the past several years, we have improved the flash photolysis-resonance fluorescence (FPRF) technique sufficiently to be able to obtain a precision of better than 3 % in measurements of 

the kinetics of OH reactions over

the temperature interval of 

atmospheric interest, and have 

applied this capability to

determine more accurate and 

reliable atmospheric lifetimes. In the past year, we carried out an extensive review of the

determination of atmospheric lifetimes, with particular reference to measurement methods and 

techniques for OH kinetics and procedures for estimating the ODP and GWP. The review covered 

all aspects of atmospheric lifetimes, from the basic concept of an atmospheric loading, removal

processes, sources and estimates of OH, and how atmospheric lifetimes are determined and 

used. The major part involved a critical review of the accuracy and precision of all commonly 

utilized techniques and a thorough analysis of uncertainties and its proper treatment. Finally, the

evaluation of OH reaction rate constant data was discussed in detail. The review treated in great

detail areas that are often glossed over, such as uncertainty analysis. Thus, it is expected to be

relied upon heavily in subsequent data analysis efforts. Although discussion was particularly 

aimed at OH kinetics, the review is clearly applicable to kinetic measurements of other radicals 

and will have an impact well beyond its immediate target. 

The accurate measurements of OH rate constant over the wide temperature range using FP-RF 

technique will be continued for selected reactions. Firstly, reliable data are needed for particular 

compounds of industrial interest. Secondly, a number of new studies of OH reactions are being

done using a relative rate techniques at room temperature, which require reliable and accurate

absolute data for the rate constants of reference reactions. Thirdly, accurate measurements of 

temperature dependences when being combined with the higher temperature measurements 

create an experimental basis to support developing calculation tools for predicting the reactivity of 

new chemicals. 

Collaboratory for Multi-Scale Chemical Science 

T. C. Allison (838); L. Rahn, (Sandia National Laboratory) 

This project is part of the DOE's National Collaboratories 

Program which funds initiatives to create collaborative

infrastructure in a variety of scientific fields. The purpose of

the Collaboratory for Multi-Scale Chemical Science (CMCS) 

project is to enable groups of scientists to work together more

productively; collect relevant data and archive it in a 

searchable manner; provide an alternate venue for scientific

publication; foster communication between scientists in different disciplines; and reduce the time 

and effort required to attack large problems. 

CSTL scientists will 

continue to work with DOE 

on CMCS for another one to 

three years depending on 

funding. 

The CMCS project is creating a collaborative environment for 

research in the chemical science areas. This environment is

centered around a web portal. It was necessary to create the

infrastructure that would provide useful tools to create, collect, 

analyze and share data. It must also offer the means to annotate data, creating new and open

formats for various data and its associated pedigree. Finally, it must establish a framework in 

which scientists from different disciplines working on problems with dramatically different length

scales can work in a meaningful and productive fashion. 

To that end, a simple object access protocol (SOAP) service was established which makes a

large database of chemical names, synonyms, formulae, and CAS numbers accessible within the

68
CMCS portal. Critical components of the CMCS infrastructure related to 

searching were extensively modified to enhance response time and make the

infrastructure extensible to larger data collections. A set of recommendations 

for attaching pedigree information to chemical data was developed. At the

present time a great deal of the infrastructure has been created and the focus is 

shifting toward demonstration of the capability of the CMCS framework in 

addressing real scientific problems. Near term activities will include the development of more

powerful searching techniques, development of "universal" formats for relevant types of data,

creating translators for legacy data, and further incorporation of NIST data accessible through the

CMCS portal. 

Weakly-bound Isomers of the ClOO Free Radical 

K.K. Irikura (838) 

The principal gas-phase cycle for the catalytic destruction

of stratospheric ozone involves the chloroperoxyl radical,

ClOO. Surprisingly little is known about this important 

molecule. For example, its structure has not been 

determined experimentally. One recent experiment,

however, suggests the existence of an isomer of ClOO 

that is very weakly bound. The atmospheric implications

of this finding, and even its veracity, were unknown. 

A recent experiment provided 

evidence for the existence of a 

weakly-bound form of the ClOO 

radical, which is important in the 

stratospheric destruction of 

ozone. The primary goal of this 

work was to verify or deny the 

existence of such an isomer. 

Recent theoretical work here at NIST has confirmed the

experimental result and determined that the Cl-O bond

in this isomer is 20 times weaker than in the stable form 

of ClOO. The potential energy functions for three of five

excited valence states of ClOO have been mapped. 

The figure shows a contour plot of the potential energy 

function. Furthermore, the experimental inference of 

weak binding has been corroborated for two of the 

three excited states that were mapped for potential 

energy. The secondary goal of this project is to

characterize the new states for spectroscopic 

identification. Further calculations are underway to

characterize the weakly-bound states sufficiently well 

for their implications in atmospheric chemistry to be

determined and for the experimental spectrum to be

assigned. 

In addition to their potential environmental impact,

these results are theoretically important for two reasons. First, this may be representative of a 

more general feature of the gas-phase chemistry of oxygen. Second, the type of electronic 

structure represented by this radical isomer is especially challenging for popular methods of 

quantum chemistry, thus providing a good test case for the development of better methods. 

Detailed dynamics calculations must be done to

determine whether the new forms of ClOO are

important in atmospheric chemistry. Spectroscopic 

simulations should also be done for comparison with

the experimental observation. Related systems, such

as ONOO, should also be investigated to learn if 

weakly-bound isomers are a general feature of the gas-phase chemistry of oxygen.

This study on ClOO chemistry

provides important data for global 

ozone models to predict the impact 

of the ClOO radical in the upper 

atmosphere. 

69
SRMs for Air Particulate Matter Less Than 2.5 μm (PM2.5) 

R. Zeisler, J.R. Kucklick, B.J. Porter, D.L. Poster, M.M Schantz, R.O. Spatz, and S.A. Wise 

(839) 

The Environmental Protection Agency (EPA)

issued new national ambient air quality 

standards in 1997 for air particulate matter (PM).

The new standards will allow regulation of the

respirable PM fraction, that is, PM <2.5 μm 

(PM2.5 or fine PM). The new standards 

accompany existing standards for PM <10 μm 

(PM10). Research recommendations have 

already been made by the NRC at the request of 

Congress and EPA to focus on evaluating the 

types of particles that cause detrimental health effects. To support compositional analyses and

other investigations on the fine PM, quality assurance 

materials are necessary. Since few appropriate reference

materials are available, NIST and EPA have an agreement

to develop, certify, and issue a suite of SRMs that will be

used for the evaluation of analytical methods to provide

national measurement traceability and to improve interlaboratory comparability of PM2.5 measurements. 

In cooperation with USEPA, NIST is

establishing benchmark SRMs for 

monitoring elemental, organic carbon, 

and other species in air particulate 

matter that will be used to evaluate 

analytical methods, help to provide 

measurement traceability, and improve 

inter-laboratory comparability of PM

measurements.

 

Newly released SRM 2783, air 

particulate on polycarbonate 

filters, is intended for elemental 

analysis using X-ray fluorescence, 

neutron activation analysis, and 

other analytical techniques. 

SRM 2783 provides 0.5 mg of fine PM on a

polycarbonate filter membrane with certified 

mass loadings for 24 toxic and environmentally 

important elements. The material for the SRM

was collected in Vienna, Austria as total 

suspended PM and then processed by the 

International Atomic Energy Agency to resemble 

a PM2.5 distribution. 

For organic compounds in PM2.5, we are in the

process of collecting a 200 g bulk sample of fine

PM at a site in Baltimore, MD with a high-volume

sampler (figure). Because the collection,

preparation, and certification of this SRM will 

require 2 to 3 years, an interim reference

material was prepared from a 20 g batch of PM2.5 

collected earlier at the Baltimore site during different sampling periods in 1998-99 and 2001. This 

interim reference material has been characterized at

NIST for polycyclic aromatic hydrocarbons (PAHs) and

nitro-substituted PAHs and has been distributed to 18

laboratories as part of a NIST/EPA interlaboratory 

comparison for the determination of organic 

compounds. Based on these results NIST will value 

assign analyte concentrations and make the material 

available to the broader community.

Cyclone sampler (left) and filter carriers

(right) for collecting large amounts of PM. 

The results of this intercomparison 

will be used with NIST measurements 

to assign concentration values for the 

compounds of interest. The interim 

reference material will then be made 

available to laboratories involved in 

EPA’s PM2.5 research programs. 

70
Development and Evaluation of Analytical Method for Nitro-substituted Polycyclic 

Aromatic Hydrocarbons in Atmospheric Particulate Matter 

M.M. Schantz, H.A. Bamford, and S.A. Wise (839) 

Nitrated PAHs (nitro-polycyclic aromatic 

hydrocarbons) are often present on 

ambient atmospheric particles from either 

direct sources, such as diesel and gasoline

exhaust or gas-phase reactions of PAHs

with nitrogen oxides. An analytical method

has been developed using pressurized 

fluid extraction followed by isolation of the 

nitro-PAH fraction from the parent PAH 

fraction using normal-phase liquid

chromatography (LC) with final analysis using gas chromatography/mass spectrometry in the 

negative chemical ionization mode. Using this method, 28 mononitro- and dinitro-PAHs were

quantified in two NIST air particulate reference materials, SRM 1648 Urban Particulate Matter 

and SRM 1649a Urban Dust. In addition, nitro-PAH 

determinations were made on a fine air particulate 

matter (particle size <2.5 μm) that was collected in

the late 1990s as part of the NIST/EPA effort to 

develop a fine particulate matter reference

material. 

Using a selective fractionation and analytical 

method, nitro-PAH concentrations were 

determined in existing NIST SRMs of 

atmospheric particulate matter as well as a 

more recent collection of fine atmospheric 

particles. These newly analyzed materials 

will aid in assuring the accuracy of results 

from monitoring studies of nitro-PAHs in 

ambient air.

While nitro-PAHs are present in air 

particulate samples in the low ng g-1

range, they are persistent in the 

environment and have been shown to be 

more mutagenic and carcinogenic in 

some cases than the parent PAHs.

Relative Abundance (SIM m/z 247)

SRM 1649a Extract, SPE only

SRM 1649a extract, SPE and LC separation

34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 min

4-NP

8-NF

1-NP

2-NP

2-NF

The normal-phase LC fractionation step was 

critical for the correct determination of nitroPAH concentrations in air particulate

samples. The figure compares the GC/MS 

chromatogram of the nitrofluoranthene and 

nitropyrene molecular ion (m/z of 247) before 

and after extracts of SRM 1649a were 

fractionated using the LC method. Prior to LC 

fractionation (top chromatogram), there are a 

large number of interfering peaks; whereas, 

after LC fractionation, quantitative analysis of

the nitrofluoranthenes and nitropyrenes is 

possible. In addition, the use of a moderately 

polar GC column resulted in the separation

and quantification of some isomers that coelute on the typically used non-polar GC 

columns. For example, when using a nonpolar GC column the most prominent peak above, 2-nitrofluoranthene, which is prevalent in 

ambient air, co-elutes with 3-nitrofluoranthene, which is prevalent in diesel emissions. 

71
Developing Methods for the Determination of Past-Use and Current-Use Persistent 

Organic Pollutants in Standard Reference Materials 

J.R. Kucklick, M.M. Schantz, S.A. VanderPol, K.J. Tuerk, and S.A. Wise (839) 

For many persistent organic pollutants 

(POPs) such as polychlorinated biphenyl 

(PCB) congeners and chlorinated 

pesticides, well-established measurement

methods and reference materials are

available for several different matrices.

However, for some pollutants such as

toxaphene, polychlorinated naphthalenes 

(PCNs), and polybrominated diphenyl 

ethers (PBDEs), established analytical 

methods or natural-matrix certified

reference materials are lacking because 

these compounds are either more challenging to measure or they have only recently emerged as 

compounds of environmental concern. For example, toxaphene is listed in the Stockholm 

Convention on POPs for elimination, while PCNs are candidates for elimination. PBDEs are a

class of fire retardant compounds whose concentrations are increasing exponentially in humans

and are being extensively monitored and investigated. To meet the need for reference materials, 

we have developed methods and provided reference values for these compounds in selected 

SRMs.

Reference values for the PCNs, 

toxaphene, and the PBDEs on relevant

environmental matrix SRMs will provide 

laboratories a tool to help assess data 

quality. SRM 1944 and SRM 1649a were 

chosen as interlaboratory comparison 

materials for the Second International

Intercomparison Exercise for the PCNs, 

which is administered both by NIST and 

Environment Canada. 

Toxaphene and selected toxaphene congeners were 

measured in SRM 1945 (Organics in Whale Blubber), 

SRM 1946 (Lake Superior Fish Tissue), and SRM 

1588a (Organics in Cod Liver Oil) using gas 

chromatography/mass spectrometry with negative

chemical ionization (GC/MS-NCI). Average total 

toxaphene values (figure) for SRM 1588a were in 

excellent agreement with a prior interlaboratory study 

using this material. Average values in SRM 1946 

were at the Food and Drug Administration’s 

Consumption Advisory Limit (2 mg kg-1), while 

toxaphene levels in SRM 1945 were about 50

% of this value. The five major PBDE 

congeners present in most biological samples

were also measured in SRMs 1588a and 1945

using GC/MS with electron impact ionization.

The levels are in a useful range for routine

environmental analysis and agreed well with 

measurements made on these two materials by 

other laboratories. PCN congeners were 

measured in selected abiotic SRMs including

SRM 1944 New York/New Jersey Waterway 

Sediment, SRM 1649a Urban Dust, and SRM

1941b Organic in Marine Sediment. PCN 

Levels were determined in the SRMs using

GC/MS-NCI following isolation using a liquid

chromatography technique that isolates planar

compounds. 

SRM 1945

SRM 1588a

SRM 1946

Total Toxaphene (mg/kg) 0

1

2

3

4

5

6

7

SRM 1588a

Interlaboratory Study

Fish SRMs

Whale Blubber

 SRM

NIST has developed methods for a wide range 

of persistent organic pollutants, including 

PCBs and chlorinated pesticides, for assigning 

concentrations in environmental matrix SRMs. 

The compounds studied here include the 

banned pesticide toxaphene, polychlorinated 

naphthalenes (PCNs), which are mainly used 

in the electrical industry, and polybrominated 

diphenyl ethers (PBDEs), which are currently 

used as flame-retardants. 

72
Expansion of NIST’s Environmental Specimen Banking Activities 

P.R. Becker, R.S. Pugh, S.S. Vander Pol, J.R. Kucklick, R.D. Day, S.J. Christopher, B.J. 

Porter, M.B. Ellisor, and S.A. Wise (839) 

NIST maintains the National Biomonitoring 

Specimen Bank (NBSB) both on the NIST 

Gaithersburg campus and at the recently 

completed Hollings Marine Laboratory in 

Charleston, South Carolina. Established in 

1979, the NBSB is a cryogenic environmental 

specimen bank that stores specimens such as 

human livers, human diet samples, fish 

tissues, mussels, oysters, and marine 

sediments collected as part of several 

monitoring and research programs supported 

by a number of federal agencies. 

With the completion of its new cryogenic 

specimen banking facility at the Hollings

Marine Laboratory in Charleston, SC,

NIST is expanding its capability to support 

monitoring and research on marine

environmental health issues. The banking

of marine mammal tissues (blubber, 

kidney and liver) is a major effort; 

however, additional programs have been 

added including the banking of bird eggs 

and feathers and marine mammal blood. 

All of these programs have been 

established at the request of other agency 

sponsors, i.e., National Oceanic and Atmospheric Administration (NOAA), U.S. Geological Survey 

(USGS), and U.S. Fish and Wildlife Service (USFWS), and with the requirement that NIST

develop collection and banking protocols for the new matrices. 

Current projects at the Hollings Marine Laboratory include the National Marine Mammal Tissue

Bank sponsored by the NOAA National Marine Fisheries Service and USGS. In 1999, NIST

established egg collection and banking protocols and began to archive the contents of seabird

eggs for the Seabird Tissue and Archival Project (STAMP). This project is a collaborative effort 

among NIST, USGS, and USFWS to monitor contaminants in eggs of seabirds of the Alaska

Maritime National Wildlife Refuge. In 2002, NIST began banking blood for NOAA’s Marine Mammal

Health and Stranding Response Program. Collection 

and storage protocols were developed for whole

blood, plasma, and serum as part of health 

assessment studies on populations of bottlenose 

dolphins in the Gulf of Mexico and on the US Atlantic 

coast. Marine mammal blood is now being banked at 

the HML facility. The latest addition to banking has 

been an effort begun in 2003 to bank peregrine 

falcon eggs and feathers for the USFWS. 

The STAMP egg collection has resulted in the 

banking of contents from 321 eggs representing 

three species of seabirds (common and thick-billed murres, and black-legged kittiwakes) from 

colonies in Alaska. NIST recently analyzed banked

tissues from 47 white-sided dolphins from the

Atlantic coast and 15 rough-toothed dolphins from 

the Gulf of Mexico for persistent organic 

contaminants and heavy metals. The contents of 67

murre eggs from colonies in Alaska have also been 

analyzed for persistent organic contaminants and

mercury. 

The largest number of specimens in the 

NBSB are from marine mammals with

Gaithersburg and Charleston together

containing 2,182 tissue specimens from 

784 individual animals representing 35

species from the Atlantic and Pacific

oceans, Gulf of Mexico, and Alaska. 

73
SRMs for Contaminants in Marine Sediment and Aquatic Species Tissue 

S.A. Wise, S.J. Christopher, R. Demiralp Oflaz, R.R. Greenberg, J.R. Kucklick, S.E. Long, 

E.A. Mackey, B.J. Porter, D.L. Poster, M.M. Schantz, and R. Zeisler (839) 

The certification of several sediment and 

tissue matrix SRMs has been completed in 

the past year including two renewals of 

previous SRMs and two new materials.

Both marine sediment SRM 1941b and 

mussel tissue SRM 1974b have certified

concentrations for 60 polycyclic aromatic 

hydrocarbons (PAHs), polychlorinated 

biphenyls (PCBs), and chlorinated

pesticides compared to only 11 and 9 

certified constituents in the original 

sediment and mussel tissue SRMs issued in 1989 and 1990, respectively. 

Natural matrix SRMs consisting of sediment 

and aquatic species tissue have recently 

been developed to address the need for 

natural matrix SRMs in assuring quality of 

environmental measurements for inorganic 

and organic contaminants. These materials 

include marine sediment (SRMs 1941b, 2702 

and 2703), mussel tissue (SRM 1974b) and 

fish tissue (SRM 1946 and SRM 1947).

A new marine matrix SRM,

2702 Inorganics in Marine

Sediment, was issued last 

year along with SRM 1946

Lake Superior Fish Tissue.

SRM 1946 is the first NIST

fish tissue SRM and is 

intended for both the 

environmental measurement

community with certified 

values assigned for 30 PCBs,

15 chlorinated pesticides, total mercury, and methylmercury, and the food 

and nutrition measurement community with certified values assigned for 

proximates and 13 fatty acids. Both SRM 1946 and SRM 1974b are issued as frozen tissue 

homogenates rather than freeze-dried tissues to provide a matrix similar to that typically analyzed

in analytical laboratories. A second fish tissue (SRM 1947 Lake

Michigan Fish Tissue) has been prepared and measurements are in

progress to provide a fish tissue SRM with emphasis on the trace

element content to complement SRM 1946, which focuses primarily 

on the organochlorine contaminants. 

SRM 1941b Organics in Marine Sediment 

and SRM 1974b Organics in Mussel Tissue 

(Mytilus edulis) are the third issue of these 

popular marine matrix SRMs for organic 

contaminants, which have found 

widespread use in a number of marine

monitoring programs supported by the 

Environmental Protection Agency (EPA) 

and the National Oceanic and Atmospheric 

Administration (NOAA). 

SRM 2702, which replaces the popular 

SRM 2704 Buffalo River Sediment, has 

certified values for 25 elements,

reference values for 8 elements, and 

information value for 11 elements 

covering all of the priority pollutant 

elements. In addition, the stock 

material used for SRM 2702 is also

being used to develop the first

reference material (SRM 2703 Marine Sediment for 

Microsampling) for solid sampling direct analysis techniques 

that commonly use small analytical test portions.

74
Detoxification of PCB-Contaminated Materials using Electron Beam Technology and 

Ultraviolet Radiation 

D.L. Poster (839); P. Neta and R.E. Huie (Div. 838); O. Kantoglu, J. Silverman, and M. AlSheikhly (Univ. of Maryland) 

The widespread use of chlorinated organic 

compounds such as polychlorinated biphenyls 

(PCBs) in a range of applications challenges the 

environment because of the toxicity of these

compounds, their persistence in the environment,

and the deleterious effect of their bioaccumulation 

in organisms and aquatic sediments. Reduction or 

removal of chlorinated organic contaminants from 

environmental systems is complex. Most materials 

that contain chlorinated species are incinerated. This method, however, suffers from two 

disadvantages: the medium is incinerated along with the contaminants, and some contaminants 

may be converted to other toxics such as dioxins. Degradation of chlorinated species by radiolytic

or photolytic methods potentially overcomes these disadvantages. However, when applied to 

organic-rich compartments such as sediment, the process can be inefficient. Other constituents in

sediment compete or react with secondary electrons 

and organic radicals produced during radiolysis. We

have investigated procedures to suppress these 

competing processes while at the same time enhancing

the dechlorination yield.

For several years, CSTL has 

collaborated with the University of 

Maryland on studies of the radiolytic 

and photolytic dechlorination of 

polychlorinated biphenyls (PCBs). 

New results show that aqueous-based

additives enhance the dechlorination 

process. 

The feasibility of electron beam 

treatment for PCB-contaminated 

systems has been demonstrated and

initial design parameters for possible 

scale-up have been established. 

500 1500 2500

dose (kGy)

% dechlorination 50

100

gamma irradiations ( Co) 60

electron beam irradiations

In earlier work, PCBs in water and

transformer oil were successfully

dechlorinated using electron beam and 

gamma irradiation technology (figure), but 

more recently, we have moved to the more

complex matrix of marine sediment. The 

use of aqueous-based additives (e.g., 

alcohol solutions and triethylamine) to

enhance dechlorination processes with

electron beam and ultra-violet 

technologies has effectively degraded 

PCBs and a metabolite of the chlorinated

pesticide DDT (DDE) in marine sediment 

(SRM 1944). In the electron beam 

irradiated samples, the concentrations of 29 PCB congeners decreased with dose (83 % at the

highest dose). Photolysis alone led to little dechlorination, but photolysis with added base led to

about 60 % dechlorination. Current investigations focus on the dechlorination of contaminants in 

the presence of food-grade surfactants. Initial results suggest that these additives enhance the 

electron beam and ultra-violet induced destruction of PCBs in marine sediment. 

Measurement of Ultra Low Sulfur in Fuel Products for Next Generation Internal 

Combustion Systems 

R.D. Vocke and W.R. Kelly (839) 

Fuel sulfur is the principal poison affecting the sophisticated catalytic converters and on-board

diagnostic systems that are central to reducing pollution from advanced internal combustion 

technologies. As the requirements for reducing these emissions from internal combustion engines 

become more stringent, extremely efficient and long-lived after-treatment systems are being

75
nationally and internationally mandated by 

regulatory actions. Through a staged process, 

petroleum producers are seeking to produce 

gasoline and diesel fuels that are functionally

“sulfur-free” by the end of this decade. In 

support of these efforts, we have produced two 

new low sulfur fuel SRMs and one near-zero 

sulfur fuel SRM that will help producers meet

current and evolving regulatory limits. 

The United States has called for a limit of 

15 μg g-1 sulfur in diesel fuel by 2006 and 

a limit of 30 μg g-1 S in gasoline by 2007. 

SRM 2723a and SRM 2299 should fully 

meet metrology needs for sulfur 

regulation of diesel fuel and gasoline, 

respectively, at these levels. 

We have completed the characterization of the sulfur content of two low-S fuels and one nearzero-S fuel. Certificates have been issued for SRM 2299, Sulfur in Gasoline (Reformulated), and

SRM 2723a, Sulfur in Diesel Fuel Oil, which have sulfur mass fractions of 13.6 μg g-1 ± 1.5 μg g-1

and 11.0 mg kg-1 ± 1.1 mg kg-1, respectively. Depending on the applicable national emission

standard, near-zero or sulfur-free means a maximum S concentration between 5 μg/g and 10

μg/g. Such near-zero sulfur levels pose significant analytical challenges. SRM 2298, Sulfur in

Gasoline (High Octane), was however, certified for a sulfur mass fraction of 4.7 μg g-1 ± 1.3 μg g1

. Capitalizing on recently improved analytical capabilities stemming from lower sulfur blanks and

improved blank control, we are completing work on another near-zero sulfur fuel SRM (Sulfur in

Kerosene) whose relative total uncertainty has been reduced by more than an order of magnitude

from that reported for SRM 2298. In addition to

developing sulfur SRMs, we are the pilot 

institution for an International Committee of

Weights and Measures-Consultative

Committee on the Quantity of Material 

(CCQM) Key Comparison and Pilot Study 

aimed at assessing participating national 

metrology institutes’ ability to provide accurate

measurements of low and very low sulfur diesel

fuels. We anticipate that, at the conclusion of 

the CCQM Key comparison and Pilot Study, we

will have another diesel fuel SRM with sulfur 

mass fraction near 40 μg g-1. 

Nitrogen oxides and non-methane organic 

gases are the primary emissions from

internal combustion engines that impact 

ozone and particulate matter pollution. To 

reduce these emissions, “after-treatment” 

systems to further remove these engine 

gases are being nationally and internationally

mandated by regulatory action. Sulfur is the 

most deleterious “poison” to these systems 

and impedes the implementation of all major 

air pollution reduction strategies in the 

transportation sector. 

International Infrastructure for Traceability in Atmospheric Ozone Measurements 

P.M. Chu and J.E. Norris (839) 

NIST is pursuing several efforts to support 

the growing national and international 

interest in the comparability and SItraceability of global O3 measurements.

NIST is partnering with BIPM to develop 

advanced primary O3 reference standards 

and to share the dissemination of

secondary or transfer standards. The basic 

project plan includes 1) Transfer the current 

O3 “national reference photometer” 

technology to BIPM. 2) Assess the

performance of the current standard reference photometer (SRP) with respect to current and 

future O3 measurement requirements. 3) Develop a primary reference standard with capabilities

of SI-traceability verification. 

The concentration of ozone (O3) in the 

atmosphere remains a significant issue from 

the perspectives of health, science, and 

politics. Collaboration with BIPM along with 

19 other National Measurement Institutes 

and the development of the advanced 

primary ozone reference standard will 

substantially improve the SI-traceability of O3

measurements.

76
Over the past two years, NIST 

has been working extensively 

with BIPM to transfer the

responsibility of international O3

measurement traceability to 

BIPM. This effort has included

the construction of five new 

SRPs, including extensive

training of BIPM staff during the 

construction process. This work completes a major portion of NIST’s collaboration with BIPM to

transfer the current SRP capabilities to BIPM. As an initial effort to evaluate the current level of 

international comparability of O3 standards, NIST is participating, along with 19 other National 

Metrology Institutes in the CCQM-P28 pilot study, led by BIPM. During FY 2003, a gas phase

titration experiment was performed to check the consistency of the current SRP O3 measurement 

capability with NIST NO gravimetric standards and an NO2 permeation calibration system. The

results indicate the O3, NO and 

NO2 standards agree to within 

1 %. Under NIST’s Small

Business Innovative Research 

(SBIR) Phase II funding, 

Aerodyne Research Inc. is 

developing a quantum cascade

laser system to serve as NIST’s 

authoritative O3 reference 

standard. Aerodyne’s

successful Phase I results have 

demonstrated that this new 

technology will significantly

extend NIST’s measurement

capabilities for O3, NO and NO2. 

At tropospheric levels, O3 is a health concern and 

contributes to global climate change as a greenhouse gas, 

while stratospheric O3 protects earth from harmful UV 

radiation. Since 1983, NIST has provided the infrastructure

for the calibration and traceability of O3 measurements 

through the distribution of Standard Reference Photometers 

(SRPs) based on UV photometry to US Environmental 

Protection Agency (EPA) facilities and 16 laboratories 

abroad.

Demonstration of real-time quantum cascade laser

measurements of ambient O3 and NO by Aerodyne Research

Inc., SBIR Phase I funding. 

Development and Measurement of Heavy Hydrocarbon (C6-C16) Gas Standards 

G.C. Rhoderick (839) 

Due to the health risk associated with long-term 

exposure to diesel exhaust from vehicles and

stationary equipment, it was requested that NIST

develop measurement standards containing heavy 

(C10 – C16) alkanes, which could then be used to 

assure measurement quality in air monitoring and 

exhaust studies. In response, a number of primary 

gravimetric standards were developed and analyzed

by gas chromatography (GC) with a flame-ionization

detector (FID). 

CSTL researchers have developed a 

number of primary gravimetric 

standards for heavy alkanes to 

address measurement needs of 

state and federal agencies that 

monitor ambient air for compounds 

related to diesel exhaust. 

While the development of C6-C10 compounds as standards was highly successful, C11-C16

compounds proved more challenging because of difficulties in retrieving these higher-boiling

compounds from gas mixture cylinder for analysis. Standards were prepared gravimetrically and

intercompared using GC-FID. Taking into account safety issues, system temperatures were

elevated to the maximum levels including the FID at 350 °C and the sample transfer system from

77
the gas cylinder to the GC column at 250 °C. GC-FID results agreed very well with the gravimetric

values, differing within ± 0.6 % for all hydrocarbons. However, analyses using varying system

temperatures exhibited major differences. 

GC-FID response factors should be the same for 

each hydrocarbon regardless of system 

temperature since the FID signal is proportional to

the number of carbon atoms detected per unit

time. The figure, however, shows that except for 

hexane, response factors at 250 °C are larger

than at 22 °C. These results suggest that the

heavier hydrocarbons absorbed within the 

regulator and the sample transfer line from the 

cylinder to the GC. However, results show a 

consistent response for an individual 

hydrocarbon. 

The results of this work indicate that if the 

efficiency of sample transfer from the

cylinder to the GC remains constant, one 

should be able to use a heavy alkane 

standard to determine an unknown provided 

that the carbon number of the standard

alkane is the same as the carbon number of 

the sample alkane. 

Global Climate Change Measurement Needs Development of Gas Mixture Standards to 

Support 

R.C. Myers and G.C. Rhoderick (839) 

Primary gas standards for CH4, CO2, CF4, N2O, 

and SF6 were developed to address quality 

assurance in measurements of climate change

gases. A number of specific drivers for this work 

can be identified. First, there was a need to

replenish the NIST in-house inventory of CH4 and

CO2 primary standards for supporting several NIST 

SRMs containing CH4 and CO2, which are frequently used by the atmospheric research 

community. A total of 22 gravimetrically prepared primary standards of CH4 in air, many of which

were prepared this past year, now exist and are used to define the NIST primary calibration

methane scale for laboratory measurements and traceability. This suite of primary standards 

range in concentration from 0.8 μmol mol-1 to 10 μmol mol-1. 

Primary standard were developed for 

several radiatively-active atmospheric 

gases: methane (CH4), carbon dioxide 

(CO2), tetrafluoromethane (CF4), 

nitrous oxide (N2O), and sulfur 

hexafluoride (SF6).

Second, primary standards were needed to support the NIST FTIR Spectral Database. Three 

standards each of CF4 and N2O were prepared for analysis by FTIR to determine band strength

intensities, which are to be archived in the database. 

The database is then typically used as a reference

data standard to determine chemical species 

concentrations in unknown gas samples and for 

measurements in real time. 

A number of additional NIST primary

standard materials will likely be 

developed for species that contribute to 

global warming, possibly leading to the 

development of new SRMs to support 

national ambient monitoring of those

species. Third, primary gas standards were needed for NIST’s 

participation in comparison studies with other

78
national metrology institutes (NMIs). One study with BIPM’s Consultative Committee for Amount

of Substance, CCQM-P41 Greenhouse Gases, involves several NMIs and laboratories from the

World Meteorological Organization in an effort to evaluate and improve the preparation and

measurement capability of gravimetrically prepared CH4 and CO2 mixtures. Two primary gas

standards, CH4 in air and CO2 in air were submitted by NIST for evaluation. A second BIPMCCQM study, CCQM-K15, involves CF4 and SF6. Here, a suite of three standards containing CF4

and SF6 were prepared in order to assign values to the CCQM-K15 sample. The third study is a 

comparison of three NIST primary standards of CH4 in air with primary standards prepared by the 

Climate Modeling and Diagnostics Laboratory of the National Oceanic and Atmospheric 

Administration.

79
80
6. Food and Nutritional Products 

CSTL supports the food industry by providing reference measurements and reference standards for quality assurance and to help 

ensure compliance with the nutritional labeling regulations. CSTL

provides robust metrological traceability for nutrients in food 

products, contaminants and

adulterants in food products,

detection of genetic 

modifications in food products,

and chemical composition and contaminants in dietary 

supplements/nutraceuticals. More than 50 % of the U.S. 

population uses dietary supplements, accounting for roughly 

$10 billion in sales every year. 

The U.S. processed food and 

beverage industry is a major 

participant in the global market. 

Almost half of the world’s top 50 

food processing firms are 

headquartered in the U.S.

Development of Analytical Methods and SRMs for Chemical Characterization of Botanical 

Dietary Supplements 

S.A. Wise, T.A. Butler, S.E. Long, M.C. Mildner, E.A. Mackey, K.E. Murphy, K.W. Phinney, 

B.J. Porter, L.C. Sander, M.B. Satterfield, K.E. Sharpless, L.J. Wood, and L.L. Yu (839); 

T. Ihara (National Metrology Institute of Japan, AIST). 

Ephedra sinica 

In 2001 NIST, the Food and Drug 

Administration (FDA), and the National 

Institutes of Health’s Office of Dietary 

Supplements (NIH-ODS) initiated a multi-year 

program to develop Standard Reference 

Materials (SRMs) and analytical methods for a

number of botanical and botanical-containing

dietary supplements; common examples 

include St. John’s wort, ephedra, ginkgo, and

saw palmetto. The goal of this collaborative

program is to provide SRMs for eight to ten 

different botanical dietary supplements over a

six-year period. Potential health risks may 

result from contamination (e.g., pesticides, heavy metals), adulteration (presence of unlabeled

foreign materials including pharmaceuticals), or variability in product composition (e.g., changes 

in levels of active constituents). Secondly, product quality and consistency must be maintained, 

through verification of dietary supplement label claims. These 

needs can be addressed by the development of analytical 

methods and reference materials to support chemical 

measurements for dietary supplements. FDA and NIH-ODS 

identified Ephedra-containing materials were identified as the

highest priority by. A suite of ephedra-based materials has been

prepared consisting of authentic ephedra plant material. These

materials have been ground and sieved to 80 mesh, as 

necessary, blended, packaged, and radiation sterilized for 

distribution as SRMs. Qualitative and quantitative chemical 

characterization of these five materials is nearing completion. 

The six ephedrine alkaloids, the primary active markers in

ephedra, have been measured at NIST using several analytical 

methods (i.e., liquid chromatography with UV absorbance 

detection (LC-UV), LC/mass spectrometry (MS), LC/tandem MS 

(MS/MS), and capillary electrophoresis (CE-UV) to assign concentration values. Measurements

are being done for contaminants such as toxic metals (As, Cd, Hg, and Pb) using inductively

Taxonomically authentic botanical 

reference materials with assigned values 

for active and/or marker compounds are 

needed for quality assurance of 

analytical measurements associated with 

the manufacturing process and for the 

verification of manufacturers’ label 

claims for the dietary supplement 

industry. Assigned values for 

contaminants and adulterants are also 

necessary to address public health and 

safety concerns.

81
coupled plasma MS (ICP-MS) and instrumental neutron activation analysis (INAA). Collaborating 

laboratories including FDA, National Research Council Canada, and ChromaDex are providing 

additional analyses of the ephedra SRM 

suite. An LC and thin-layer chromatography 

(TLC) profile or “fingerprint” of the plant

material will also be provided for use as a 

reference for comparison to other similar 

materials.

The next high priority botanical materials for

development of SRMs are: Ginkgo biloba, 

St. John’s wort, saw palmetto, and green tea.

The ginkgo materials (plant, extract, and 

solid oral dosage form) have been obtained

and the preparation (grinding, blending, and 

bottling) of this SRM suite is in progress. Acquisition of the

St. John’s wort, saw palmetto, and green tea are in progress 

and will be completed in early 2004. NIH-ODS also plans to

expand the scope of this project to include multivitamin and 

multielement supplements. The development of a

multivitamin and multielement dietary supplement SRM, with

values assigned for the 35 vitamins and elements listed on

the supplement label, will be a major effort in the next two

years. 

NEW SRMS PRODUCED IN FY 03 

SRM 3240 Ephedra sinica Stapf (aerial parts of

authentic plant) 

SRM 3241 Ephedra sinica Stapf (dried native

extract) 

SRM 3242 Ephedra sinica Stapf (dried

commercial extract) 

(dried extracts either natural or fortified to 

contain 8 % total ephedrine alkaloids) 

SRM 3243 Ephedra-Containing Powdered Solid 

Oral Dosage Form (finished product) 

SRM 3244 Ephedra-Containing Protein Powder

(finished product) 

The availability of SRMs with 

certified concentrations of 

active/marker constituents and 

contaminants provides the 

measurement tools necessary to 

assess the quality of dietary

supplements. 

Certification of SRM 1946 Lake Superior Fish Tissue – Frozen Fish Tissue for 

Environmental Contaminants and Food Constituent Measurements 

S.A. Wise, S.J. Christopher, J.R. Kucklick, S.E. Long, E.A. Mackey, C.S. Phinney, B.J. 

Porter, R.S. Pugh, D.L. Poster, M. Rearick, KE. Sharpless, and M.M. Schantz (839) 

NIST natural matrix SRMs for organic and inorganic 

contaminants in marine tissue are necessary for the 

development and validation of analytical procedures and to

provide quality assurance of chemical measurements in the

marine environmental and food measurement communities. 

SRM 1946 Lake Superior Fish is a fresh frozen tissue

homogenate prepared from trout collected in Lake Superior 

and was developed primarily for use in the determination of

organic contaminants with certified and reference concentrations for 42 polychlorinated biphenyls 

(PCBs) and 17 chlorinated pesticides, which is the largest number of organochlorine compounds 

with values assigned in any natural matrix SRM 

or in any certified reference material (CRM) from

other national or international sources. SRM 

1946 has values assigned for 13 PCB 

congeners that have not been previously 

measured in any natural matrix SRMs. This is 

the first natural matrix SRM with certified values 

for the three non-ortho-substituted PCB 

congeners, which are present at 10 to 100 times lower concentrations than the other PCB 

congeners measured, but they are considered to be the most toxic congeners with respect to

mammalian cells and biological systems. The increased number of PCB congeners with 

assigned values in this SRM was a result of improvements in the analytical methodology used for 

SRM 1946 Lake Superior Fish 

Tissue, is the first fish tissue 

SRM, and is intended to 

address the needs of both 

the environmental 

contaminants and food 

measurement communities. 

82
these measurements, particularly for the non-ortho-substituted PCB congeners, and the 

methylmercury. 

The development of SRM 1946, with certified 

and reference values for over 100 chemical 

constituents, including 3 coplanar PCB 

congeners and the omega-3 fatty acids, 

represented a significant technical challenge. 

This SRM is the only CRM of its kind in the 

world and will serve as an invaluable quality

assurance tool for quality assurance of 

contaminant measurement programs related to 

the marine environment on local, regional, and 

continental scales.

Because SRM 1946 is comprised of edible fish tissue, the food chemistry community can also

use this material for measurements that are necessary to support nutritional labeling

requirements. SRM 1946 is characterized for nutrient concentrations including proximates (fat,

protein, carbohydrates, ash, and solids), 25 individual fatty acids (the most fatty acids measured

in any SRM), and 12 nutritive minerals and trace elements. The individual fatty acids that are 

certified in SRM 1946 include the nutritionally important omega-3 fatty acids, which have not been

certified previously in a natural matrix SRM. 

Of particular importance in both the 

environmental contaminants and food

measurement communities is the 

determination of total mercury and the more

toxic mercury species, methylmercury. Both

of these environmentally relevant species are 

characterized in SRM 1946. This is the first 

certification of these compounds in a fresh

fish tissue homogenate CRM. The certified 

concentration of methylmercury in SRM 1946 is approximately at the level proposed by the U.S.

EPA for freshwater and estuarine water quality criterion (0.3 mg/kg fish). Thus this SRM will be

valuable in assessing methods used to monitor methylmercury levels in fish to determine

allowable fish consumption advisories. 

A companion fish tissue material, SRM 1947 Lake Michigan Fish Tissue has been prepared and 

measurements are in progress to provide a fish tissue SRM with emphasis on the trace element 

content to complement SRM 1946, which focuses primarily on the organochlorine contaminants. 

SRM 1947 will be completed in FY04. 

 Publications: “Determination of Polychlorinated Biphenyl Congeners and Chlorinated 

Pesticides in a Fish Tissue Standard Reference Material,” Poster, D.L., Kucklick, J.R., Schantz,

M.M., Porter, B.J., Leigh, S.D., and Wise, S.A. Anal. Bioanal. Chem., 375, 223-241 (2003). 

“Determination of Non-ortho Polychlorinated Biphenyls in Environmental Standard Reference 

Materials,” Brubaker, Jr. W.W., Schantz, M.M., and Wise, S.A., Fresenius’J. Anal. Chem., 367, 

401-406 (2000). 

 “Determination of Methylmercury and Butyltin Compounds in Marine Biota and Sediments 

using Microwave-Assisted Acid Extraction/Digestion, Solid-Phase Microextraction, and Gas 

Chromatography with Atomic Emission Spectrometric Detection,” Tutschku, S., Schantz, M.M., 

and Wise, S.A., Anal. Chem., 74, 4694-4701 (2002). 

A Complementary Suite of Food-Matrix SRMs to Support Industry Compliance with 

Nutritional Labeling Requirements 

K.E. Sharpless, J. Brown-Thomas, C.S. Phinney, B.J. Porter, and L.J. Wood (839)

The composition of a food-matrix 

SRM should be matched closely to

that of the test sample being

analyzed; a fat-protein-carbohydrate

food composition model developed by

AOAC International is a useful way to 

do that. NIST has been working with 

other federal agencies and the food 

CSTL researchers develop reference materials and

analytical methods to underpin measurements 

made for compliance with nutritional labeling 

laws, provide traceability for food exports needed 

for acceptance in many foreign markets, and 

improve the accuracy of information that is 

provided on product labels to assist consumers in 

making sound dietary choices. 

83
industry over the past several years to provide a suite of SRMs within this model to address

nutrition-labeling issues. Two new food-matrix SRMs were completed this year: SRM 2385

Slurried Spinach and SRM 2387 Peanut Butter. Now, there are food-matrix reference materials 

with values assigned for nutrient concentrations available 

within all sectors. Ideally, these materials will 

improve the accuracy of nutrition information 

provided on product labels. In the figure, all 

of the food matrix SRMs are shown in their 

respective positions in the AOAC triangle. 

Now that materials are available within or on the borders of 

eeping Tin Cans Lead-Free

S.E. Long (839)

hen steel mills manufacture tin-plated sheet

3 Pig Tin, with a

veloped in

p

all sectors, this particular aspect of NIST’s food and

nutrition program can be expected to decrease. Focus will 

then shift to more specialized analytes (e.g., individual

amino acids, allergens, mycotoxins) in foods, as well as to 

dietary supplements. 

K

J.R. Sieber, K.E. Murphy, and

W

steel for food cans, they must test the tin and the

electroplating bath for lead content. If lead is 

allowed to accumulate in the tin or the bath, it will 

become part of the tin coating and may leach into 

food stored in cans. In recent years, the allowable 

Pb content of tin plate has been reduced in

recognition of potential health risks in products 

used for foods.

Steel mills typically 

purchase Sn anodes 

according to ASTM B 3

restriction of 50 μg g

9 Standard Specification for

-1 Pb maximum. Residual amounts of anode tin 

are typically remelted on-site to form new anodes. Incoming and

remelted tin anodes are analyzed by atomic emission spectrometry 

to meet material acceptance and process control requirements. 

SRM 1727 Anode Tin was de

cooperation with ASTM International to provide 

a tool for improving the accuracy of Pb

determinations at trace levels in tin, tinplate

products, and plating bath solutions. SRM 1727

is certified for Pb content on the basis of

analyses by isotope-dilution inductively-coupled

ectrometry. Information values are provided for 

ten additional elements. SRM 1727 is sold in solid form (a block 

30 mm on each side) for spectrometric analysis and may be 

chipped for use with chemical methods of analysis. 

plasma mass s

Tinplate is a high volume commodity 

rs

made by many US and foreign steel 

companies. About 3 million tons of 

tinplate are manufactured and 

shipped from US steel produce

annually, at a typical selling value of 

$800 per ton during 1999. Container 

manufacturers purchase much of the

tinplate to make products such as 

food and beverage cans. 

SRM 1727 will provide a 

e

se 

ovide 

means for validation of th

accuracy of analytical 

determinations of Pb at

trace levels enabling the

industries to control their 

tinplate lines more 

economically and pr

safer products. 

84
Plasmid-Based Standard for Quantitative Real Time Polymerase Chain Reaction (PCR) 

dard was 

i no known

round-robin intercomparison pilot study was set up

Measurements 

M.J. Holden and J.R. Blasic, Jr. (831)

CSTL researchers are investigating the utility of a

synthetic DNA construct for the validation of

measurement protocols, equipment, personnel and

varied laboratory practices used in the quantitation of

specific DNA sequences. This cross-platform material 

will provide information on the comparability of different 

platforms and detection probe types. 

The material for this study and future stan

designed from the ground up so that it could be used

with different types of protocols, instrumentation and 

probes. It was also des

gene. The designed DNA sequence (200 base pairs) was 

synthesized by a self-assembly process and ligated into a

plasmid. The plasmid was sequenced, propagated in

bacteria, isolated and purified on cesium chloride gradients.

PCR protocols were designed and assays conducted to verify 

the suitability of the material for this purpose. Studies were 

conducted to determine the recoverability and stability of the

plasmid material after lyophilization. 

by the Comité

gned so that it resembled

Quantitative Real Time PCR is 

s

a powerful tool for the 

detection and quantitation of

specific DNA sequences. It 

has broad applications in area

including: biotech crop 

measurements, health care,

pharmaceutical development

bio-warfare agent detection, as

well as basic research.

,

The material designed by

A

Consultatif pour la Quantité del Matière (CCQM) under the auspices of the

Comité International des Poids et Mesures (CIPM) and the Bureau

International des Poids and Mesures (BIPM). The CCQM has the 

participation of 14 laboratories from around the world including national 

measurement institutions from 11 countries and the European Union. Multipl

DNA materials were shipped to the participants. Test materials were prepared so that

participants in the study could produce a standard curve of known DNA concentration and then

analyze and report on the values of DNA samples of unknown concentrations. The participants 

could select their instrumentation and probe type and were free to design whatever measurement

protocol they deemed suitable. Several of the laboratories utilized more than one probe type and

instrument. Very recently, the intercomparison participants returned their data and detailed

information on how the studies were conducted. Analysis of the data is now underway. 

he results of this study will provide basic information on the comparability of instrument and 

CSTL researchers is being

used for the validation of 

measurement protocols,

equipment, personnel and

varied laboratory practices

applied to the measurement

s

of

pecific DNA sequences. 

e sets of lyophilized

T

detection platforms for quantitative real time PCR. It will also shed light on sources of error and

point to ways to improve and standardize methods. The study will lead to the improvement of the

design of the material itself and clarify the utility of this type of material as a standard for

monitoring the quality of quantitative PCR measurements. It is likely that the plasmid material 

will be redesigned to accommodate detection platforms that have been recently developed. 

85
86
7. Forensics and Homeland Security 

CSTL has provided DNA standards for human identification since 1992;

however, over the past year the effort has 

focused on new methods of analysis to

respond to the need to analyze extremely 

degraded samples. CSTL has also had a long 

history of assessing standards needs and

developing new measurement technologies for crime scene 

investigations in which handguns or explosives are used. The

forensic community both for quality assurance and to provide legally

defensible analyses uses reference standards. CSTL is also active

in the development of microsensor arrays and other measurement

technologies for the identification of explosives, as well as chemical 

and biochemical weapons. CBRNE (Chemical, Biological, Radiation,

Nuclear, Explosives) related measurements, methods, and standards is a major new area of

expansion for CSTL. 

Particle Removal Rates from Planar and Cloth Surfaces; A Critical Step for Providing 

Airport Security 

R.A. Fletcher, G.J. Gillen (837), and E. Ferguson (SURF Student) 

In a portal detection system, an object or person is 

subjected to an air jet array that dislodges and 

transports explosive particles to an ion mobility 

spectrometer for identification and detection. The

ability to remove particles from various surfaces is the

critical first step in successful detection of trace 

explosive residues. CSTL researchers are developing

the methodology to determine particle removal rates 

from various surfaces likely to be encountered in an

airport screening environment. We are interested in the conditions necessary 

to optimize remove of particles of various sizes and compositions. 

CSTL researchers are assisting 

the Transportation Security 

Administration (TSA) in the 

evaluation of next generation 

walk-through portal explosive 

screening devices to support 

security checkpoint screening at 

US airports. 

To date we have assembled a system to test 

gas jet removal of particles from surfaces. With

this system, we utilize monodisperse

fluorescent polymer spheres ranging from 1 μm to 45 μm in

diameter as test particles distributed on two materials;

polycarbonate surfaces and muslin cloth. In this initial set of 

experiments, we are using spherical particles in an effort to verify 

our experimental approach before studying more complex 

particles (high explosives) and surfaces. After particles are

dispersed on the substrate, optical 

images of the sample surface are

taken with a fluorescence microscope

using 488 nm laser illumination. A 

high velocity, computer controlled,

nitrogen gas jet is used to remove the 

particles from the surface. 

Micrographs are taken before and 

after exposure of selected areas of 

the surface to the gas jet. Particle 

removal rates from the respective

Commercial walk-through 

portal for explosive 

detection. 

87
surfaces are determined by particle counting 

implemented through image processing and

analysis. The micrographs shown in the figure

at right illustrate typical before and after images

and subsequent identification of the particles 

removed by the air jet exposure. Below is a 

plot of the particle removal efficiency as a

function of jet pressure for particles of different 

sizes on a polycarbonate surface. Here the 

measured jet pressure relative to ambient lab 

room pressure (Pjet/Patm ) is plotted against the

percentage loss of particles. The pressure ratio

is proportional to the gas velocity (and other 

fluid dynamic parameters) and for Pjet/Patm < ca 

1.8, the jet is subsonic. The figure

demonstrates that the particle removal

efficiency is strongly dependent on particle size.

Particles in the range of 6 μm to 10 μm in

diameter and smaller (1 μm particle data not

shown) are tightly bound to the surface and are 

not effectively removed by our pulsed air jet system. Since the size of explosive particles in trace

residues (such as fingerprints) appears to be in the micrometer and smaller range, further 

optimization of the air jet removal process is needed for effective sampling of these materials. 

Micrograph of 20 μm and 40 μm Fluorescent 

Spheres on Polycarbonate Before and After 

Air Jet 

An overlay of these 

two images (inset) 

demonstrates

identification of

removed particles 

(light blue). 

Before After

The work described here may help improve the design of next-generation explosive detectors by

defining optimal parameters for air jet removal of particles. The metrology capabilities that we 

have implemented will also be used to test new and 

improved methods for particle removal including the

use of ionized air and CO2 gas jet systems. As next

steps we plan to increase the reliability of the 

measurement by automating both data acquisition 

and image analysis. 

Plot of particle loss from polycarbonate filters for 

45 μm (circles), 20 μm (squares) and 6 μm to 10 μm 

polystyrene spheres (triangles) as a function of gas 

jet pressure relative to ambient pressure. A 

quadratic fit was made to the data. 

0

20

40

60

80

100

1 2 3

Percent Loss (%)

Pjet/Patm

45 μm 

20 μm 

6-10 μm 

Particle Size Characteristics of Trace High Explosives: Pentaerythritoltetranitrate (PETN) 

and Cyclotrimethylenetrinitramine (RDX) 

J.R. Verkouteren (837) 

The need to screen for explosives at 

airports and other points of entry has

resulted in the deployment of thousands of

trace explosives detectors and created a 

critical need for effective calibration of 

these detectors. The detection of traces of 

high explosives is often dependent on 

collection of explosive particles, particularly 

for low vapor pressure explosives such as 

RDX and PETN. Of fundamental importance in determining the effectiveness of any particle 

CSTL researchers in collaboration with the 

Transportation Security Administration 

(TSA), the NIST Office of Law Enforcement 

Standards (OLES), and the NIST Advanced 

Technology Program (ATP), are investigating 

particle size in explosive traces to develop 

effective methods and materials for testing 

and calibrating trace explosives detectors.

88
collection scheme is the size and distribution of particles expected in the traces left by handling 

explosives. 

Particle collection can be accomplished by many methods, including swiping of surfaces, 

agitation with air jets, etc. The ability to model particle size in explosive traces was tested by

measuring the particle size distributions produced by fragmentation of crystals of RDX and PETN. 

These particle size distributions were found to deviate from a power law (linear under a log

transformation), probably due to a tendency of the explosives, particularly PETN, to smear rather

than fragment. The data indicate that more small particles are expected to result from 

fragmentation of RDX compared to PETN, which matches observations of particle sizes in

fingerprints produced from C-4 (a compound containing RDX) and Semtex-A (a compound

containing PETN). 

To simulate the particle size produced during handling of explosive compounds, crystals of RDX 

and PETN were removed from the explosive compounds C-4 and Semtex-A, respectively, and 

crushed to produce populations 

of particles that were sized using 

image analysis and scanning

electron microscopy. In addition,

fingerprints with the two 

compounds were simulated by 

handling C-4 and Semtex-A and

successively pressing a finger on

a series of glass slides for

analysis by polarized light

microscopy (PLM). The figure 

shows that the deviation is more 

pronounced for PETN, with

fewer numbers of smaller 

particles expected than for RDX.

These data agree with PLM 

observation of the fingerprints,

as more small particles of RDX 

are observed, compared with 

PETN, although the RDX 

particles tend to be bound by the 

plastic matrix. Tenth generation

fingerprints of Semtex-A contain 

many wide (up to 100 μm 

diameter) thin plates of PETN,

consistent with a smearing 

process. 

log d, μm

1 10

log n

100

>d 1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

PETN

RDX

Plot of the particle size distributions of RDX and PETN 

particles determined by scanning electron microscopy. 

The size distributions deviate from the expected power 

law (linear under a log transformation) resulting from a 

fragmentation process probably due to the tendency of 

the explosives, particularly PETN, to smear rather than 

crush.

Working with TSA, OLES, ATP, and with first 

responder organizations, our results will be

applied to the development of test materials 

and methods used to determine the 

effectiveness of trace explosive detectors.

Knowledge of the particle size of explosives in

trace residues, particularly as they differ for

different compounds, will guide the

development of challenge samples and sampling schemes. The ultimate goal is to improve

detection of explosives residues and therefore support Homeland Security efforts. 

The results obtained from

this work will be used to 

guide improvements in 

explosive particle 

sampling procedures for

trace explosive detection 

equipment. 

89
Adsorption of Energetic Materials (Explosives) on Surfaces 

T.J. Bruno (838); K.E. Miller (Univ. of Denver)

The development of surrogate soil stationary 

phases for gas chromatography was pioneered

at NIST for the evaluation of contaminant 

adsorptive interactions in the environment. We

have now extended our approach to the liquid

phase by producing heat-treated spray dried soil 

surrogates, and have applied these surrogates 

to the study of energetic materials (explosives), 

residues and taggants. The application of our 

adsorption metrology to energetic materials is 

important because of the increased emphasis 

on homeland security. Several new detectors have been developed for in-the-field detection of

explosives, but these devices cannot be used until their performance is certified. Certification

cannot be done without an explicit knowledge of the vapor phase concentration of the explosive

being measured. Since surfaces play a significant role in determining how much material will 

exist in the vapor phase, an understanding of the adsorptive interactions is critical. The

chromatographic approaches that we have developed are particularly apt in providing that

understanding. 

Because of the critical need to detect 

the presence of explosives, residues, 

taggants, etc., in a variety of venues 

(public places, military installations, 

hostile locations abroad), several highly

sensitive detectors have been 

developed. Before these devices can 

be used in most such applications, their

performance must be certified. 

Following our earlier development of a method to prepare clay soil surrogates (suitable for gas

chromatographic adsorption enthalpy measurements), we have now extended the technique to

the liquid phase. This was done by preparing a surface by spray-drying a clay suspension to 

produce 8 μm to 10 μm spherical particles that were subsequently heat-treated at 500 ◦

C. This

material was then slurry- packed in an HPLC column configuration. Measurements were done on

a series of substituted benzenes (including hetero-substituted benzenes such as RDX) to

determine the partition coefficient, retention factor and enthalpy of adsorption, ∆Hads, in the

presence of water. Our measurements led us to a predictive correlation of the interaction on the

basis of substituents on the main ring. Our correlation allowed us to conclude that the interaction

of substituted benzenes on clay is remarkably similar 

to that on porous graphitic carbon. At the same time, 

we noted dramatic differences when the interactions

were compared on an organic-like surface such as 

octadecyl silane. In addition to the development of this 

predictive correlation, we have measured the enthalpy 

of adsorption of energetic materials on the clay surface

in the presence of water. We found, for example, that 

the ∆Hads of RDX is 30 % higher than that of benzene

on the clay. We have also completed measurements 

of the enthalpy of solution, ∆Hsol, of a number of 

energetic materials in a polymer, polydimethylsiloxane. 

Thus, the ∆Hsol of TNB, TNT, and RDX were measured 

to be 57.9 kJ/mol, 59.5 kJ/mol, and 62.4 kJ/mol, 

respectively. It is interesting to note that these values are approximately 20 kJ/mol lower than the 

enthalpy of vaporization, ∆Hvap, for these compounds. 

This work has demonstrated the 

importance of surfaces when

considering the free concentration of

energetic materials that need to be

detected in the field, and the need to 

have a predictive capability for a 

variety of compounds. Our 

observations of the similarity of the 

clay surface with graphitic carbon, 

and the enthalpy difference between

the bulk solid and absorbed 

explosives will advance our ability to 

certify new detection devices. 

Development of SEM-EDS Analysis Procedures for C, N And O to Enable Characterization 

of Explosives Particles, Polymeric And Organic Materials 

J.T. Armstrong and J.A. Small (837) 

Details provided in the Exploratory Research Section 

90
Determination of Traces of Fissionable Materials Using Delayed Neutron Activation 

Analysis 

R.M. Lindstrom, E.A. Mackey, and G.P. Lamaze (839) 

Detection and measurement of small traces of

fissionable uranium and plutonium can be done 

by delayed neutron activation analysis. The 

method is intrinsically specific to nuclear fission, 

the sensitivity is excellent, and the procedure is 

simple, rapid, and readily automated for high

throughput. 

IAEA inspector collecting a swipe sample from 

process equipment in a nuclear fuel fabrication 

facility in 1994 (Anal. Chem., 74, 28A, 2002). 

Delayed Neutron Activation Analysis is being 

established at NIST for the measurement of small quantities of fissionable nuclides such as 235U 

and 239Pu. After a brief neutron irradiation, the sample is 

placed quickly into a neutron detector array and the neutron

emission rate measured and compared with that of a 

standard. The method is well-tested, rapid, specific, matrix 

independent, nondestructive, and sensitive. The system

being built at NIST is calculated to have a detection limit for 

either of these species about 10 pg, based on a 

straightforward extrapolation from published practice. The 

analysis time is less than 2 min per sample. 

An important tool in nuclear forensics 

is the collection and analysis of 

“swipe” samples at sites where 

materials of interest may be, or may 

have been at one time. Tiny traces of 

fissionable uranium or plutonium can 

be left behind whenever these materials 

are handled or transported. 

Preliminary tests have been performed using uranium 

standards prepared by depositing solutions containing 

known amounts of uranium onto filter papers, irradiating,

and counting on a detection system with one 3He detector 

surrounded by hydrogenous moderator. Two shielding 

configurations were tested, one using water 

as the moderator and the other using

polyethylene. Based on these results and a 

review of the literature, a final detection

system was designed. This consists of ten neutron detectors in a 30 x 30 cm cylindrical 

moderator of polyethylene, lined with 2 cm of lead to absorb gamma radiation. The design of the 

manual system incorporates the existing pneumatic rabbit assembly for irradiation control. 

Incorporated in its design is the ability to unload a sample from the receiver after irradiation with

compressed air. This feature will be used to move the 

sample rapidly to the neutron detector through a 

polyethylene flight tube. 

In FY 04, we plan to improve sample throughput, the

transfer system can be readily automated with computer 

control. It has been demonstrated elsewhere that 233U, 

235U, and 239Pu can be distinguished by the relative yields 

of delayed neutron precursors with different half-lives, and 

of several fission products. We plan to add a gamma-ray 

detector into the neutron moderator to exploit this 

signature. 

This research will provide the 

Nation with a readily accessible, 

rapid means of measuring traces 

of fissionable U and Pu in 

samples of forensic interest. In 

addition, the specificity and 

sensitivity of this method of 

analysis will be put to use in 

certifying trace uranium in 

Standard Reference Materials. 

Mapping the Protective Antigen Binding Site on the Anthrax Toxin Receptor 

K.D. Ridge (831) 

Details provided in the Exploratory Research section.

91
Automated Analysis of Organic Particles Using Cluster SIMS

G.J. Gillen (837)

Trace analysis of explosive and narcotic 

residues is important for homeland security, 

customs, forensic, law enforcement, and 

environmental applications. Key issues of

research for these applications include the

nature of the trace residues to be sampled

(particle size distribution of explosives and

narcotics in fingerprints, for example), the 

efficiency of various types of particle collection

systems, and the development and 

characterization of organic particle standard reference materials. Each of the areas requires the

use of analytical techniques that can rapidly and uniquely 

identify micrometer-sized explosive/narcotic particles

among a background of ambient particles. We are 

evaluating secondary ion mass spectrometry (SIMS) for this 

application. Automated imaging analysis of organic

particles by SIMS has not previously been feasible due to

the sensitivity limitations imposed by low molecular

secondary ion yields, degradation of the sample by the 

primary ion beam and the prohibitively long acquisition 

times that are required to survey multiple fields using low

primary dose SIMS conditions. However, recent research

suggests that the use Cluster SIMS, which provides large

enhancements in molecular ion yield and a reduction in 

beam-induced damage may overcome the previously 

mentioned limitations, making organic particle searching

feasible. In the present study, we explore the uses of SIMS 

particle searching, combined with energetic cluster ion

bombardment using SF5

+

 or C8

- primary ions, to rapidly 

characterize the distribution of explosive and narcotic 

particles on various surfaces to support continued

development of trace detection screening technologies for

homeland security. Characteristic SIMS mass spectra were obtained from particles (a few 

micrometers in diameter) of various pure 

explosives and narcotics dispersed on silicon 

wafers using either SF5

+

 or Cx

-

 (x=1-10) primary 

ion bombardment. As expected, cluster

bombardment provided large enhancements in

molecular secondary ion signals compared to 

monoatomic bombardment under identical 

conditions. For example, C8

-

 bombardment gave characteristic molecular ion signals for

explosives that were 300 to 1000 times greater than for C1

-

 bombardment under identical 

conditions. All of the samples examined also exhibited significant reductions in beam-induced

damage under cluster bombardment. The combination of high secondary ion yields and reduction

in beam-induced damage allows us to image the distribution of organic particles while achieving 

high count rates and short acquisition times. SIMS particle searching is currently being 

successfully employed in our laboratory to determine the distribution of explosive particles in

fingerprints, the efficiency of different types of explosive particle collection filters and to study the

release of particles during the sampling process. 

CSTL researchers explored the uses of 

SIMS particle searching, combined with 

energetic cluster ion bombardment to 

rapidly characterize the distribution of 

explosive and narcotic particles on 

various surfaces to support continued 

development of trace detection screening 

technologies for homeland security. 

Mosaic of secondary 2.5 mm ion microscope images of acetaminophen 

(m/z = 152). Image acquisition 

time 20 s. SF5

+

 primary ion 

bombardment with beam dose 

during acquisition of 2.3x1013

ions/cm2

. 

Cluster beam SIMS offers increased 

secondary ion signals and the capability

for minimizing beam-induced damage

and therefore allows for the rapid 

characterization of organic particles on

surfaces. 

92
Certification of NIST Reference Material Supporting Improvised Explosive Device 

Measurements 

W.A. MacCrehan and M. Bedner (839) 

As part of their investigations of explosives 

crime, national, state and local forensic

laboratories measure the additives in

smokeless powder (gunpowder). US military 

laboratories also measure the chemical 

composition of such propellant powders to 

determine the stability of munitions.

Improvised explosives devices (IEDs), such as

pipe bombs, most commonly have smokeless 

powder as the active filler ingredient. When

investigating an explosives incident, forensic investigators collect residues of the incompletely 

burned powder from the crime scene. As part of the evidence investigation 

process in the forensic laboratory, the physical appearance and chemical 

composition of the powder are carefully measured. This permits a

categorization of the residue powder for comparison to samples of known

powders. The resulting information can often be used to link residues to

powders recently purchased by or collected in evidence from the 

perpetrator. 

Smokeless powder is a blend of 

nitrocellulose and a series of additives 

including the propellant nitroglycerin 

(NG) and stabilizers such as 

diphenylamine (DPA) and ethyl centralite 

(EC, N, N’-diethyl, N, N’-diphenylamine) 

along with N-nitrosodiphenylamine 

(NnDPA) that forms during aging of DPAstabilized nitrocellulose. 

To help assure the quality of forensic measurements 

of evidence such as explosives residues, the 

forensic community has a certification program for 

laboratory measurements, called ASCLD/LAB 

(American Society of Crime Lab 

Directors/Laboratory Accreditation Board). As 

part of a five-year certification process,

laboratory methods and operator 

proficiency are tested with challenge

samples of known properties. Currently, there are no test or 

reference materials to support explosives measurements. To 

address the need for forensic explosives reference materials, the

NIST Office of Law Enforcement Standards (OLES, in conjunction 

with the National Institute of Justice) and the NIST SRMP have

sponsored the development of RM 8107 Additives in Smokeless Powder. In addition to the need 

of forensic laboratories, precise measurement of the chemical composition of smokeless 

propellants is critical to the US military community as a means of assuring the safety of munitions. 

Certification of NIST RM 8107 will promote accurate forensic and military smokeless powder

measurements. 

In 2004 NIST will release 

RM 8107, Additives in 

Smokeless Powder thus

providing a reference 

material to support the 

quality of forensic and 

military smokeless powder 

measurements.

RM 8107 consists of a rifle-type ball powder. A major powder 

manufacturer, Primex Technologies, provided 4 kg of rifle powder from a

single, carefully blended manufacturing batch. For the certification 

measurements of the four additives, an ultrasonic solvent extraction method was developed to

recover the analytes. Two separation techniques were used to determine the NG, DPA, NnDPA,

and EC content: micellar capillary electrophoresis and liquid chromatography (LC). The mean

values obtained by the two techniques were in good agreement. Since the LC technique 

provided lower uncertainty in the measurements, it is being used for the certification

measurements. 

93
With the development of this smokeless powder reference material, forensic laboratories will have

a reliable material for the development and validation of measurement methods for improvised 

explosives devices. The reference material may also be used as a proficiency challenge sample

to test operator and laboratory performance in explosives measurements as part of laboratory 

accreditation activities. In addition, US military laboratories can use the additive certified

reference values in the NIST reference material to assure accurate monitoring of munitions 

stability. 

The smokeless powder RM 8107 was developed to help assure the quality of ‘low explosives’ 

type measurements. However, ‘high explosives’ such as TNT and RDX (ingredients of military 

and plastic explosives) are often used in terrorist explosive 

devices. In conjunction with NIST’s OLES and the DHS’s Office 

of Domestic Preparedness, we plan to develop a reference 

material for these high explosives. A solid matrix material will be 

coated with commonly used military explosives. The goal is to develop a particulate explosives

material that can be used to test explosives detection equipment used for airport security

screening, evaluation of suspicious packages by first responders, and forensic laboratory postblast investigations. 

Refractive Index Measurements of FBI Liquid for Forensic Glass Particles 

J.R. Verkouteren (837) 

The FBI uses a light-microscope-based heating 

stage technique for refractive index 

measurements, and disseminates reference 

materials and training to hundreds of crime labs 

throughout the country. The measurement

technique requires that the glass particles be

immersed in a liquid, the refractive indices of which 

are known for wavelengths spanning the visible 

range, and for temperatures ranging from ambient

to 100 °C. The success of litigation often hinges on

the accuracy of these measurements; therefore the FBI requested calibration of their immersion 

liquid through the Office of Law Enforcement Standards (OLES) using a highly precise technique 

developed in CSTL. 

Microscopic glass particles play a

major role in forensic analysis aimed 

at linking individuals to crime scenes. 

Refractive index can be used to prove 

a common source of glass particles, 

and is readily measured from 

microscopic particles using relatively 

inexpensive equipment. 

The refractive indices of the FBI liquid were

measured at four wavelengths in the visible

and at three temperatures ranging from 20 °C 

to 65 °C using the method of minimum 

deviation (see figure). The data were modeled to provide 

equations to calculate refractive index at any temperature 

from 0 °C to 100 °C and at any wavelength in the visible.

The uncertainties are an order of magnitude larger than the 

level requested by the FBI owing to limitations of the liquid 

itself. The FBI liquid is very viscous and optical homogeneity

was compromised, probably as a result of persistent

temperature gradients on the order of a few hundredths of a

degree C. As the light microscope technique used by the

FBI depends on temperature stability of the same order for 

optimum refractive index accuracy, our results indicate that

this liquid is probably not the best choice for a reference liquid. 

Crime labs throughout the 

country collecting refractive

index data on glass particles 

must know the accuracy of the 

technique in order to support 

their results in the courtroom. 

Our work with the FBI will help 

to establish the uncertainties of 

the method by working to 

establish appropriate reference 

materials. 

94
The work described here established the 

need for a new immersion liquid. Therefore,

NIST’s Physics Laboratory, in collaboration

with CSTL will produce a glass standard

reference material as a calibration material 

for the immersion technique. With both a

glass SRM and a calibrated liquid, crime 

laboratories will be able to determine the

uncertainties of the refractive index 

immersion technique. 

T, °C

10 20 30 40 50 60 70

RI

1.51

1.52

1.53

1.54

1.55

1.56

643.8 nm

508.6 nm

480.0 nm

467.3 nm

Plot of the refractive indices of the FBI 

immersion liquid provided to NIST for

calibration. The sample was measured at 

4 wavelengths in the visible and at 3 

temperatures ranging from 20 °C to 65 °C 

using the method of minimum deviation. 

NIST Support of the CDC Chemical Counter-Terrorism Laboratory Network 

K.E. Murphy, M.M. Schantz, B.A. Benner, Jr., T.A. Butler (839) 

Cyanide is considered a likely candidate for 

use in chemical terrorism because it is easy 

to obtain and has been used in previous 

attacks. Cyanide is analytically challenging,

and because it is unstable, blood-based

standards are not commercially available.

NIST developed an improved method for 

the determination of cyanide in whole blood 

based on headspace gas chromatography 

with mass spectrometric detection (GC/MS), 

employing isotopically labeled cyanide

(K13C15N) as the internal standard. Use of

the isotopically labeled internal standard coupled with GC/MS resulted in decreased analysis 

times and improved accuracy and precision. The improved method has been adapted by CDC for 

transfer to state laboratories within the CTLN and is being used to provide

value assignment and stability testing data for a pool of cyanide-spiked blood 

samples produced by CDC for the CTLN. 

NIST has developed an improved method 

for the determination of cyanide 

concentrations in whole blood samples 

and is collaborating with the Emergency 

Response and Air Toxicants Branch of the 

Centers for Disease Control and 

Prevention (CDC) to help provide quality 

assurance and measurement proficiency 

standards for the "Chemical CounterTerrorism Laboratory Network" (CTLN). 

At the start of this project, NIST did not have an inhouse method for the determination of cyanide. A 

method based on headspace analysis with GC/MS 

detection was implemented and improved by the use of

an isotopically labeled internal standard. An 

interagency agreement with CDC has been established 

to evaluate the content and stability of cyanide in 

spiked, frozen human whole blood. NIST has started a 

series of periodic analyses that will proceed over the

upcoming year to provide value assignment and

monitor the stability of the cyanide concentrations in

the proficiency standards. 

A commercial supplier has prepared 

proficiency standards containing 

different levels of cyanide and 

approximately 700 vials per level have

been packaged. Preliminary results 

indicate that the cyanide 

concentrations in all levels of the 

frozen proficiency standards have

remained stable for the past two

months. 

95
It is imperative that measurements made in

response to a chemical terrorism event be both 

rapid and accurate. There are currently no

standards to underpin the accuracy of blood 

cyanide measurements. NIST’s value assignment

and stability monitoring of these blood based

cyanide proficiency standards will enhance CDC 

and state public health preparedness in the event 

of a chemical terrorism incident involving cyanide. 

Future Plans: NIST will monitor the 

stability of cyanide in the frozen human 

whole blood proficiency standards over

the upcoming year and will continue to 

work closely with CDC to meet the needs 

of the CTLN, which has grown from the 

original five members to include 

laboratories in 46 states across the U.S. 

Cell-Based Sensors for Screening Toxins 

L. Locascio and J. Travis (839); A. Suggs, B.J. Love, and N.G Love (Virginia Polytechnic 

and State Univ.) 

This collaborative effort to develop a cell-based sensor 

has focused on the detection of electrophilic chemicals in

water since for many years Love’s group has studied the

negative biological response of bacterial cells to this class 

of toxins. In the presence of electrophilic toxins, E. coli

cells rapidly efflux a large amount of potassium into the 

surrounding media, a response that contributes to 

bacterial deflocculation and ultimately bioreactor failure.

Prototype bioreactor devices are designed as microfluidic 

chips containing immobilized bacteria with integrated optodes to detect potassium efflux upon

exposure to electrophiles. We are targeting the device for the detection of toxins in wastewater 

streams but also hope to extend this work to screen for toxins in drinking water. 

CSTL researchers and 

collaborators work to develop 

a cell-based microfluidic 

biosensor that can serve as an 

early warning system for the 

detection of chemicals in the 

wastewater that may cause 

bioreactor failure. 

Contamination of the bioreactor, the heart of the wastewater treatment plant, from chemical

dumping can have a devastating impact on wastewater processing. In fact, system shut-down 

caused by bioreactor failure often results in the diversion of large amounts of raw untreated

sewage to rivers and streams with an obviously detrimental effect. Several different classes of

chemicals, including electrophilic toxins, feeding into water treatment plants have been known to 

cause bioreactor degradation leading to system processing failures. Recent results from Love’s 

group have led to the hypothesis that sludge deflocculation or 

biofilm detachment, a mechanism by which the bioreactor can 

fail, occurs through the activation of the glutathione-gated K+

efflux (GGKE) system stimulated by electrophilic toxins.

Therefore, prior to biofilm detachment, bacterial cells expel large

amounts of potassium from inside of the cell to the outside. In

this collaborative effort, we have designed a cell-based 

microfluidic biosensor to detect the presence of electrophilic 

toxins in water streams entering wastewater treatment plants in

an effort to prevent failure of the bioreactor. The approach is to immobilize bacterial cells in a 

microfluidic system and monitor cell response, in particular potassium efflux, as the water flows

through a small bed of immobilized cells. In essence, we have created a micro-bioreactor. The

behavior of our micro-bioreactor is then used to predict the behavior of the process bioreactor 

when exposed to the same water supply. A negative response in the microsystem can be used

to decide when to divert part of the incoming stream to prevent process failure. The

microbioreactor has several key components including an immobilized bed of live bacterial cells 

(Escherichia coli K-12); an optode film that is responsive to potassium ions; an optical detection

unit; and microfluidic channels to move cell media and the water samples through the cells and

past the optode film. 

In FY 03, we focused our 

attention on two aspects 

of this project: 

(1) cell immobilization in 

polymer microchannels, 

and (2) development of a 

miniaturized optical 

detection component.

96
Love’s group hypothesized that cell adhesion to an abiotic polymeric surface would be influenced

by several factors including the chemical characteristics of the bacterial membranes; the

the characteristics of the polymer

material used to fabricate

microfluidic channels. Earlier

work, published in 1987 by another 

research group in the Journal of 

Microbiology, was performed to

determine the characteristics of the

bacterial membrane, specifically 

the relative hydrophobicity of E. 

coli K-12 cells in different growth 

media, that could affect cell

adhesion. Experiments showed

that cells grown in an N-limited 

medium (20:1 C:N) exhibited 

greater cell surface hydrophobicity

than a balanced C:N media 

supporting previous findings

published by a research group in

the Journal of Applied Microbiology in 1999. Based on these results, polymers were chosen that

were expected to enhance cell adhesion and these were tested in cell binding experiments. E. 

coli cells were also immobilized onto polymer surfaces coated with polyelectrolyte multilayers. It 

was determined that the cells adhered better to uncoated polyethylene terephthalate glycol

(PETG) surfaces than to PETG surfaces coated with polyelectrolyte (see figure). 

physiological state of the cells at the time of adhesion; and

he detection system was miniaturized to make the device 

i a

tandards for Detection of Biological Threat Agents 

)

STL scientists are characterizing surrogates that can

T

more suitable as a prototype field device. A microfluidic 

device was mounted on a small holder and a miniaturized 

light source was coupled to the channel using fiber optics. 

Light was collected through fiber optics into a hand-held 

spectrometer (Ocean Optics) coupled to a laptop computer 

for data processing. This device is now being evaluated to 

measure the optode film response in the presence of

varying concentrations of potassium. We hope to expand th

microfluidic biosensor for detection of chemical and biological toxins in drinking water.

Fluorescence image of PETG microchannels after 

exposure to E. coli cells. Fluorescent dots indicate the 

presence of live cells. A) Uncoated PETG channel, and 

B) channel coated with polystyrene sulfonate. 

The development of a hand-held 

e

he

device for in-situ monitoring of 

environmental toxins in water 

streams could provide a reliabl

early warning system can have a

profound environmental and 

financial impact by detecting t

problem before failure occurs. 

s work to the development of

S

K.D. Cole, L. Wang, A. Gaigalas, and D. Hancock (831

C

safely be used for the testing of detection devices,

remediation technologies, and personnel training. There 

are many instances where these surrogates are

preferable to the use of the actual bacteria, viruses, and

toxins. We have identified several surrogates and are in 

the process of developing methods to fully characterize

them. We will use these well-characterized surrogates as 

model systems to study and improve the instruments and

devices used for the detection of biological threats. If

necessary, follow-up work will be performed at BSL-3 

CSTL researchers are working 

) and

t

with the Department of 

Homeland Security (DHS

other government agencies to 

determine the best ways that 

NIST can meet the needs for 

data, measurements, and 

reference materials for the

detection of biological threa

agents. 

97
laboratories that are able to handle the live agents. Additionally, the detection of biological

threats is done from environmental samples that represent serious challenges because of their 

complex and variable compositions and the possibility of interferences. 

uorescence

icroscopy, and DNA based assays such as PCR. 

e

racterized surrogates to determine

eir performance. 

nd Characterization of SRM 2395—A Human Y-Chromosome DNA Profiling 

.M. Butler, R. Schoske, P.M. Vallone, J.W. Redman, and M.C. Kline (831) 

standard, SRM 2395, used to

To date, we have identified several surrogates for the biological

threat agents that we can safely use in our laboratories. The 

surrogates include a spore-forming bacteria (Bacillus globigii), 

an RNA virus (the bacteriophage MS2), and the Ricin A chain.

Detection of biological threats currently depends upon either 

recognition by antibodies or detection of specific nucleic acid 

sequences. We are in the process of developing improved 

sensitivity and reliability of the assays. The methods we are

studying include classical microbiological techniques, flow 

cytometry of fluorescent-labeled surrogates, fl

m

CSTL scientists will continue to build strengths to characterize 

these surrogates and identify new ones. The devices and 

instruments need to b

from complex environmental samples. NIST can help

assure national security by helping to test these

devices and proving the reliability of both data and 

measurements. The surrogates will be used to study 

the devices and instruments used to detect existing as 

well as new emerging threats. These complex 

organisms and biological molecules will require

sophisticated techniques to characterize them. New 

devices and instruments that are being developed will 

also need well-cha

Bacillus globigii, crystal 

structure from PDB 

tested using realistic surrogates and 

The development of methodologies to 

characterize surrogates for biological

threats is important and challenging

because of the needs of accurac

sensitivity, and reliability. It

essentially certain that wellcharacterized surrogates will be us

for standardizing the devices and 

instruments used for the detection 

th

Creation a

Standard 

J

The Y-chromosome is passed largely unchanged

from father to son and contains genes that endow the

recipient with male characteristics. Forensic DNA 

testing laboratories, genetic genealogy companies,

and academic groups studying human history 

perform Y-chromosome testing by examining a

number of genetic markers. The ability to obtain 

consistent and reliable results between laboratories 

that share information through DNA databases is 

critical. To address this need, NIST has developed a human Y-chromosome DNA profiling 

check the accuracy of analytical methods used to

measure Y chromosome variation at common short 

tandem repeats (Y-STR) and single nucleotide

polymorphism (Y-SNP) markers. One unit of SRM 

2395 consists of six frozen tubes of DNA, each of

which contains approximately 100 ng of wellcharacterized extracted human genomic DNA. There 

are five male samples (Components A-E) and one 

 that can be

y, 

is

ed

and measurement of these threats. 

SRM 2395 is being used already in 

genetic genealogy companies such

Relative Genetics to calibrate their 

results. New Y-STR typing kits from 

ReliaGene Technologies and Prome

Corporation have used our SRM to 

as

ga 

verify the accuracy of their assay. 

SRM 2395 has been produced with 

multiple male DNA samples that 

have been characterized at common 

Y-chromosome short tandem repeat 

and single nucleotide polymorph

markers to enable calibration

results obtained with either 

ism

of

research assays or commercial kits. 

98
female sample (Component F) packaged in a single box. The female specimen may be used as a 

negative control for Y-chromosome specific assays. The Certificate of Analysis lists typing results

for 31 Y-STR and 42 Y-SNP markers commonly used in Y-chromosome testing. DNA sequencing

has been performed on 22 of the 31 Y-STRs to permit an even more extensive characterization of

Polymorphism Typing (SNP)

.C. Kline, J.W. Redman, P.M. Vallone, and J.M. Butler (831) 

A laboratories in a timely and costeffective manner. 

t

ng the 51 samples 

ould be sub-typed to 12 different SNP types with four samples being unique. 

ccessful results, and this information is now included in the technical

anual for this product.

those markers.

Sequencing results for the Y-STR marker DYS390 on SRM 2395 component

The sequence indicates that there

 E. 

are 24 repeats present with the motif of 

[TCTG]8 [TCTA]11 [TCTG]1 [TCTA]4

Evaluation and Automation of Single Nucleotide

Methodologies for Mitochondrial DNA (mtDNA) 

M

Procedures for analysis of mtDNA are laborious and 

expensive because they typically involve DNA

sequencing of more than 600 base pairs in the control 

region across hypervariable region I (HVI) and

hypervariable region II (HVII). Therefore, techniques for 

rapid screening of samples, particularly to weed out 

ones that do not match, will enable more samples to be

run by forensic DN

Roche Molecular Systems has developed a system that 

probes ten sites in HVI and HVII. Their manual process of 

hybridization and detection was adapted

format at NIST as part of beta-testing their

product. In addition, we ran 666 U.S.

population samples to evaluate the power

of discrimination for the mtDNA LINEAR 

ARRAY test. The 666 NIST samples

resulted in 282 different types, of which

185 were seen only once in our 

population. These unique samples represent 65.6 % of the types seen and 27.8 % of the people.

Several groupings of samples seem to represent specific ethnic groups. Fifty-one samples were

determined to be mitotype 1111111111 by LINEAR ARRAYs. After SNP typi

o an automated

c

An automated procedure for processing the mtDNA LINEAR 

ARRAYS using a Tecan/Profiblot hybridization robot is under 

development. Analysis of the mtDNA mitotypes/haplotypes in

128 Hispanic, 252 African American, and 286 Caucasian

population samples doubled the available population data on 

U.S. samples. We discovered that the pH of the washing buffer 

is critical to obtaining su

Mitochondrial DNA (mtDNA) is 

used in human identity testing

mass disaster reconstruction, 

missing person investigations, 

and forensic casework involving

for

highly degraded DNA samples. 

NIST provided beta-testing of a 

commercial mtDNA system, and

automation of the process.

commercial system offers

forensic laboratories doing 

human identification a rapid test

for screening samples that ma

or may not require laborThe

y

intensive full sequencing. 

LINEAR ARRAYS allow

laboratories without la

intensive sequencing 

capabilities to rapidly

borscreen mtDNA samples

m

99
A paper describing the automated LINEAR ARRAY 

processing is in preparation. We are in the process of

developing new SNP assays to increase the power of

discrimination for mtDNA. 

This figure shows the mtDNA LINEAR 

ARRAY results for 12 different DNA 

samples. These samples were

processed in an automated fashion in

a method developed at NIST as part of

beta-testing this Roche Molecular 

Systems product. 

Typing Single Nucleotide Polymorphisms Located in the Y Chromosome 

P.M. Vallone and J.M. Butler (831) 

A single nucleotide polymorphism or SNP is a single

base change in the human genome. A SNP is predicted

to occur every 1000 bases or so in the human genome.

There is interest in typing SNPs for human identity 

(forensic) purposes. More specifically, SNPs located on 

the Y chromosome are attractive typing candidates due 

to their low mutation rate and male specificity. The fact

that the Y chromosome is only found in males makes 

genetic markers located upon it useful in mixed malefemale sample (as found in a sexual assault crime). The 

low mutation rate of the Y chromosome SNPs implies 

potential for determining the geographical origin of an

individual. 

To date, few studies have been 

performed evaluating the 

frequency of known Y 

chromosome SNPs in U.S. 

populations. By evaluating the 

frequency of 50 wellcharacterized SNP sites in over

200 U.S. population samples 

we provide a useful database 

of DNA typing information.

 

Here, we used a commercial DNA typing kit 

and custom assays developed in-house to

determine the frequency of 50 Y SNPs in 2

U.S. populations (African American and

Caucasian). The two assay methodologies,

hybridization and primer extension, indicated 

full concordance for typing eight overlapping 

SNP sites. The overall success rate for typing

that samples was over 99 % for either

methodology. A pie chart summary of the data 

is illustrated in the figure. SNP frequency 

distributions for the 50 genetics markers typed 

indicated groupings that were unique to each 

population. 47 % and 23 % of the Caucasian

and African American samples fell into

haplogroup R1b respectively. 58 % of the 

African American samples were found to be in

the haplogroup E3a. It is of interest to note that

none of the Caucasian samples fell into

haplogroup E3a making it a population specific 

grouping. This information is useful in 

determining the forensic utility of using Y-SNP 

in forensic investigations. 

This figure represents the distribution of 

various Y chromosome haplogroups (Hg) 

for 2 U.S. populations. Hg R1b represents 

the majority of the Caucasian samples while 

58 % of the African American samples are in 

Hg E3a. Note there is an admixture of 23 %

of Hg R1b (major Caucasian Hg) observed 

in the U.S. African American population.

100
CSTL researchers have provided forensic 

scientists with population data on newly 

discovered genetic markers. This work supports 

companies who have an interest in developing 

new forensic testing kits. By providing typing 

data for the 50 Y-SNPs in a U.S. population a

company can select the appropriate markers that

will be most beneficial for human identity testing.

CSTL researchers have completed one of 

the first studies involving Y-SNPs 

frequencies in U.S. populations. The 

results of this study have been submitted 

for publication to the Journal of Forensic 

Science. This work was presented at the 

International Society of Forensic 

Genetics, Arcachon, France (Sept 2003). 

101
102
8. Health and Medical Technologies 

CSTL provides quality assurance for the healthcare industry by providing

the appropriate chemical and

physical reference standards, 

developing reference methods and

databases, and also by working

closely with the clinical and medical 

community to transfer the accuracy

of the highest level of metrology to high-throughput clinical laboratory 

measurements. Measurements are responsible for up to 15% of the

$1.3 trillion annual costs of healthcare in the U.S. A significant portion (25% to 30%) of these 

measurements are performed for non-diagnostic reasons, such as re-tests, error prevention, 

and detection.

Healthcare costs amount to ~ 

14% of the GDP, an estimated 

$1.5 trillion. By 2007, healthcare 

spending as % of GDP is 

projected to reach over 16%. 

HIV Protease Structural Database 

T.N. Bhat, G.L. Gilliland, V. Ravichandran, and M.D. Prasanna (831); J. Vondrasek and A. 

Wlodawer (NCI/NIH) 

The HIV Protease Database (HIVdb) contains 

experimentally determined three-dimensional 

structures of human immunodeficiency virus type 1

(HIV-1), human immunodeficiency virus type 2 (HIV-2),

and simian immunodeficiency virus (SIV) proteases 

and their complexes 

with inhibitors or

products of substrate cleavage. The structural holdings of the

HIVdb are from two sources, the Protein Data Bank and from 

academic, government and industrial laboratories that have not

deposited the coordinates in the Protein Data Bank.

Developed in collaboration with 

NIH, the HIV Protease Database is 

a web-based archive for HIV 

protease structural information 

located at 

http://srdata.nist.gov/hivdb.

The HIVdb permits the user to customize the query to retrieve

the desired information. The query interface provides the option 

to select 

entries by

the year in

which the structures have been solved,

resolution range, R Factor, strain in which 

the proteases were isolated from, inhibitor 

type, company or laboratory in which the

structures were solved, virus type and

citation or depositor author. In addition, the 

users have the option of dynamically 

structuring the report page that is generated. 

Future efforts will focus on converting the 

web server language from Active Server

pages (ASP) to Java-based server

languages for the option of incorporatin

Recently cited in Science’s 

NetWatch (“Databases: 

HIV’s Achilles Heel,” 

Science, 300, 1349, 2003),

the HIVdb provides 

macromolecular structural 

data on HIV and related 

proteases as well as data

analysis tools for scientists 

engaged in research and 

drug development in the

fight against AIDS.

g

readily available software. 

103
Standards Requirements for Systems Biology Approaches to Health Care: Mitochondrial 

roteomics

owards

set of

The mitochondrion represents a discrete subcellular organelle with a non-nuclear

g more

fe

in the 

Biotechnology Division was tailored for 

P

G.B. Vásquez and P.E. Barker (831); S. Zullo (ATP/NIST) 

The health care community, particularly th

a systems biology or proteomic approach

human health issues relate to

mitochondrial function, from the

biology of cancer development to

issues relating to homeland security 

that include bioterrorism and chemical 

toxicities. Mitochondrial proteins are

encoded by both nuclear and

mitochondrial genes, which makes the

human mitochondrial proteome a good

candidate for an integrated systems 

biology/proteomics model system. The 

human mitochondrion is central to

basic life functions for the generation 

of cellular energy, and as such is the site of key components of the biosynthetic pathways, 

cellular decision points leading to apoptosis (programmed cell death), and other important

functions.

at involved with clinical diagnostics, is moving t

to disease detection and diagnosis. A broad

Currently there is great hope in the utility of 

mitochondrial proteomics in health care, 

especially the areas of pharmacology, clinical

diagnostics, and cancer and heart disease 

treatments. A NIST-sponsored worksho

standards for proteomics, held on September 

17-18, 2002, reviewed the needs of the 

mitochondrial proteomics community in model

systems, methods, and data. Following the 

workshop was a

p on

genesis of proteomics efforts

by NIST to meet the standards requirements that

were identified. 

enome that is comprised of about 1000 or

ur

different protein species with tissue-specific 

A workshop to review of the standards needs 

of the mitochondrial proteomics communities, 

held on September 17-18, 2002, resulted in a 

road map for NIST’s efforts in proteomics.

This document outlines the how NIST should 

address the needs of the evolving proteomics 

community. This has also led to NIST’s 

involvement in international proteomics 

standards planning through the Human

Proteomics Organization (HUPO).

Additionally, a laboratory

at es.

The :

• ent of the mitochondrion as an 

• rds needs for 

• opriate 

data elements (Common Data Elements, 

CDEs) for health care proteomics.

focus of this workshop was to provide

assessm

integrated model for systems biology

studies, 

• assessment of emerging proteomics 

technologies, 

identification of the standa

proteomic applications in the clinical

diagnostics industry, and 

guidance for determination of appr

proteomics standardization work. 

OVERVIEW OF CSTL ACTIVITIES IN MITOCHONDRIAL PROTEOMICS STANDARDS AND DATA

• Development of an XML library for 2-D polyacrylamide gel electrophoresis (2-D PAGE) for 

• affects the

(htt

the storage and querying of proteomics 2-D PAGE data. (Veerasamy Ravichandran, MEL, 

and XML community) 

MitoAnalyzer, a program that allows the user to determine how a polymorphism

resulting protein in human mitochondrial DNA 

p://www.cstl.nist.gov/biotech/strbase/mitoanalyzer.html). (Barbara C. Levin) 

Human Mitochondrial Protein Database, which provides comprehensive data on 

mitochondrial and human nu

• 

clear encoded proteins involved in mitochondrial 

biogenesis and function. (http://bioinfo.nist.gov:8080/examples/servlets/index.html). 

(Veerasamy Ravichandran) 

The later works resulted in a follow-up meeting to the workshop, “Mitochondrial Proteomics: 

Data Issues and Standards”, held at NIST, November 5-7, 2003. 

104
Substrate Specificities and Kinetics of Action of DNA Glycosylases Involved in Cellular 

i o the o

D th elevat

d m

asure multiple modified bases in

damaged DNA. Our studies showed that extensive

DNA glycosylases. 

a better , thereby

to normal cells,

ew approaches to curing

olid-tumor cancers are being

rug 3-Amino-1,2,4-benzotriazine 1,4-dioxide

xploratory Research Section. 

Repair of Oxidative DNA damage 

M. Dizdar (831) 

Oxidative DNA damage is generated by free

radicals formed in living cells by normal 

metabolism and exogenous sources such as

ionizing radiations and carcinogenic compounds. 

Numerous products are formed in DNA by 

oxidative damage. Most DNA oxidative products 

are cytotoxic and mutagenic with deleterious

biological consequences. In living cells, repair 

systems exist to repair damaged DNA to sustain the genetic integrity and to prevent genomic

nset and progression of cancer and other diseases. 

ed genomic instability are associated with a number of 

ainly repaired by base-excision repair involving repair

enzymes called DNA glycosylases. In our studies, we 

used gas chromatography/isotope-dilution mass

spectrometry (GC/IDMS) because of its ability to

simultaneously me

A new approach has been dev

to measure how well the cell repairs 

oxidative DNA damage. This approach

eloped

involves a study of substrate 

specificities and kinetics of action of 

the DNA glycosylase repair enzyme. 

nstability, which may lead t

eficiencies in DNA repair wi

iseases. Oxidative DNA damage is

variability exists in the substrate specificities of numerous 

These measurements lead to

enabling the development of

methods for prevention of

diseases and development of 

new therapeutic approaches. 

This includes the

improvement of cancer

therapies by inhibiting DNA

repair in drug- or radiationresistant tumors by altering

the expression of DNA repair 

genes (which generate the 

DNA glycosylases and AP 

endonucleases shown in the 

figure). By coupling these

altered expression patterns 

with DNA repair inhibitors that

show strong selectivity in 

killing tumor cells in

comparison

GC/IDMS enables the precise 

determination of substrate 

specificities of DNA glycosylases by

identifying the modified bases that

are or are not removed from 

damaged DNA by a given enzyme. 

understanding of DNA repair mechanisms

Modulation of DNA Repair 

Inhibition of DNA repair in cancer therapy

n

s

developed. 

DNA Damage by the Antitumor D

(Tirapazamine) 

P. Jaruga (831, UMBC), M. Birincioglu, H. Rodriguez, and M. Dizdar (831); G. Chowdhury 

and K. Gates (Univ. of Missouri) 

Details provided in the E

105
Human Mitochondrial Protein Database: A Resource for Human Mitochondrial Proteomics 

. Ravichandran, P.E. Barker, G.B. Vasquez, T.N. Bhat, and G.L. Gilliland 

831); S.J. Zull (ATP) 

NA Repeat Element Mutation Frequency Measurements of the Polypurine/pyrmidine 

Kidney Disease

l

rtant for understanding the expression, regulation and evolution of

DNA. In e em rep

th eat ele

s

enome. Characterization of repeat elements will 

provide insights into human predisposition and 

susceptibility for disease. 

NA

ubmitted to Science. 

V

(

Details provided in the Exploratory Research Section.

D

Tract Contained in the PKD1 Gene of Polycystic

J.P. Jakupciak and C.D. O’Connell (831); A. Baco

The presence of repeated sequences is a

fundamental feature of genomes. Repeat

elements are the simplest form of regularity and 

analyzing repeats gives first clues to discovering 

new biological phenomena in the same way as 

repeated words give a starting point to

deciphering a script written in an unknown

language. The human genome consists of 

repeating DNA patterns of various sizes, from 

very small to very large. Although the functions 

of these repeating regions are not well 

understood, they appear impo

la and R.D. Wells (TAMU) 

More than 10% of the human genome is

composed of interspersed repetitive

elements. Already more than a dozen 

human diseases have been associ

with trinucleotide repeat elements. 

Therefore, identifying repeat elemen

ated

ts

that disrupt or contribute to gene 

dysfunction will significantly increase 

our knowledge of human disease. 

ukaryotic genomes, tand

at are still being discovered. Rep

uch as fragile X syndrome. 

eats are involved in various regulation mechanisms

ments are involved in human neurological disorders,

Our studies will provide fundamental measurements 

for researchers in industry and academia

investigating DNA mutation(s) associated with

human disease and supports efforts to understand 

the function of repeat e

Instability of polypurine/pyrimidine 

repeats has also been shown to be

associated with polycystic kidney

disease (PKD). We sought to mea

the mutation frequenc

sure 

y of the lements in the human 

polypurine/pyrimidine tract present in g

PKD and characterize the repeat

element instability. 

Our measurements

support the concept that 

non B-DNA structures, 

formed by the repeat 

elements, are located at 

the mutation sites.

Overall, the results 

indicate that human 

disease is associated

with DNA mutations

(break points) that

coincide with non B-DNA 

conformations. This work 

provides the first detailed 

insight regarding DNA

structural conformations

and their function at D

break points. The results Measurement of the break points of 16 long deletion events 

of this study have been 

s

106
High-Throughput Analysis of Telomerase by Cap

D. Atha and J. Jakupciak (831); W. Wang (NCI/NI

Clinical studies show a direct association

between telomerase activity and cells that are 

cancerous. The enzyme telomerase is expressed

in 85-90 % of all human cancers, but not in 

normal tissues. Currently, telomerase assays 

measure the small amount of enzyme activity in 

tissue lysates, urine or blood serum. The most

commonly used system for the detection a

i

H

nd

quantification of telomerase activity (TA) is the

P telome

re y

cell extracts with a percent standard d

obtained using the slab-gel based me

d from 0.21 to

.47, which is a variation of about 2 to 40 RNA molecules 

e eria for differentiating 

e

ilicon Nanoparticles as DNA Labels 

ry Research Section.

llary Electrophoresis and Real-Time PCR 

)

CR-based assay known as the

peat amplification protocol or TRAP assa

rase

.

Most of the TRAP assay systems use a slab-gel

based electrophoresis system to size and semiquantify the PCR products. We have improved the

sensitivity and reproducibility of this system using

capillary electrophoresis. However, such methods

are still low-throughput. We have developed a highthroughput system or RApidTRAP (robot-assistedTRAP). The MWG SE 4204 robot was used to

generate TRAP/PCR samples for analysis using an 

ABI 3100 16-capillary array instrument. TA

ethod on (50 to 500) cells per reaction isolated from tumor 

eviation (CV%) of 20. This was about half the variation

thod. Measurement of hTERT mRNA was made using

specific primers and probes on a LightCycler in the range 

of (10 to 7000) cells / reaction. The percent standard 

deviation (CV %) of these samples range

measurements were made using this m

4

p r 1000. This is well within the crit

b tween cancer and normal cells / tissue. 

S

V. Reipa and L. Wang (831) 

Details provided in the Explorato

Comparison of hTERT mRNA and 

Telomerase Activity (TA). The relationship

between the relative telomerase activity

(shown in bars) and the amount of cellu

hTERT mRNA (τ). The amount of cellu

hTERT mRNA (copy number) was measured 

in the A549 cells

lar

lar

in comparison to the 

telomerase activity (total peak area) 

obtained from automated TRAP/PCR 

measurements. 

The use of telomerase as a biomarke

for cancer has been hindered becau

of a lack of high-throughpu

which are essential for large cohort

studies. We are developing robotic 

high-throughput capillary 

r

se

t methods,

electrophoresis and real-time PCRbased (gene expression) methods for 

the analysis of telomerase. 

The TRAP assay was successfully

transferred from the manual to a robotic 

platform with results that were 

consistent in sensitivity and 

reproducibility. The sample handling 

system of robot-assisted-TRAP will

streamline validation of telomerase with 

increased sensitivity, reproducibility

and automation over previous lowthroughput methods.

107
Semiconductor Nanocrystal Probes for Human Me

P.E. Barker and Y. Xiao (831) 

About one third of breast tumors have

amplification of the gene for the tyrosine kinase

receptor HER2/neu. In contrast to the other breast 

cancer patients, HER2 overexpressor tumors can 

be specifically targeted by the first rational design 

drug, Herceptin (trastuzumab), a humanized

mouse monoclonal antibody that targets cells with 

high expression of the HER2 gene. Clinical testing 

for HER2 overexpressors follows two approaches,

FISH (fluorescence in situ hybridization), a gene

taphase Chromosomes 

te DNA evel,

a that

th org

quantitative measurements of the type necessary for

 DNA probes used for FISH testing could be

rateg may be more quantitative for medical 

st measuring the HER2 gene at the

nd an IHC (immunohistochemical) test

e cell surface. Most FISH tests employ

l

measures the amount of HER2 protein expressed on

anic fluorescent tags. 

Recently, an inorganic tag of the binary compound 

CdSe, has become available. CdSe and other similar

quantum dot labels are much more stable on

exposure to light, and are brighter than conventional 

organic fluors. The CdSe labeled DNA probes were

directly compared with fluorescein- and Texas Redlabeled probes, and proved to be superior for

This work demonstrates the physical 

advantages of quantum dots as labe

for human metaphase chromosomes. 

The purpose is to provide researchers

and clinical laboratories in in

and academia with alternative, stable 

and high luminosit

ls

dustry

y fluorophores for

measuring gene expression 

associated with medical and oncology 

diagnostic tests. 

HER2 gene analysis. It was demonstrated that

detected with quantum dot labels, and such a st

testing than prior approaches. 

y

Specificity of fluorophorestreptavidin detection of

biotinylated total human DNA in

metaphase chromosomes. 

Bar in panel C is 10 microns. 

(A) Control (no fluorophore), 

(B) Streptavidin-Qdot 605 detection 

of chromosome 1q12 region 

(vertical and horizontal arrows), 

(C) TexasRed-streptavidin detectio

of biotinylated DNA hybridized to 

n

1q12 (vertical arrows) 

(D) FITC-streptavidin detection of

1q12 sites (vertical arrows).

The IHC and FISH tests do not always

select the same patients for 

trastuzumab therapy for treating b

HER2 overexpressor tumors. In t

process of developing a standard

reast 

he

cell 

analyte with known HER2 levels, 

alternative fluorophores were

investigated in a model system. 

FISH FOR P LABEL NTUM 

DOTS, TEX RED AND FLUORESCEIN (HUMAN TOTAL 

DNA AS A PROBE) 

CONTROL

TEX RED

QUANTUM DOTS

FLUORESCEIN

B6 CELLS ING WITH QUA

108
Temperature Gradient Focusing for Biological Assays in Microchannel Systems 

K.M. Balss, D.J. Ross, and M.J. Tarlov (836); H. Begley (863); K.G. Olsen (Loyola College) 

Temperature gradent focusing (TGF) 

offers three significant advantages for 

capillary or microfluidic assays. First, 

targets present at trace levels can be

concentrated before the assay reaction 

to enhance sensitivity. Second, 

because TGF focuses targets to a fixed 

point, they can be held stationary for

mixing with a recognition probe 

transported by bulk flow. The facile 

mixing of reactants shortens analysis 

time. Third, probe-target binding partners formed in the assay are focused and concentrated at a

point spatially distinct from that of the focused targets resulting in sensitive, low-background 

detection. 

A fast and simple nucleic acid assay was 

designed to provide proof-of-concept for 

temperature gradient focusing (TGF) in capillary 

or lab-on-a-chip systems. The TGF approach 

described here using peptide nucleic acids to 

recognize other nucleic acids through 

hybridization could be extended to other 

biological assays such as measurement of 

protein/protein, nucleic acid/protein, or 

drug/target binding.

To demonstrate proof-of-concept, an assay for

DNA was developed using peptide nucleic 

acids (PNAs). PNA molecules contain the

same nucleobases as DNA and, thus, exhibit

similar base-pairing properties as DNA.

However, unlike negatively charged DNA 

molecules, PNAs are neutral. In the TGF 

assay, the PNA molecules serve as molecular 

recognition probes for a specific single-strand

DNA (ssDNA) sequence. The assay involves three steps. First, the negatively charged ssDNA 

targets, the analyte molecules, are focused and concentrated at a predetermined point in the

capillary. Second, fluorescently labeled PNA is then introduced in the capillary and carried by 

bulk flow of the buffer through the focused ssDNA. Third, if PNA/DNA hybridization occurs (i.e.,

the ssDNA is complementary to the PNA) the negatively charged ssDNA/PNA duplex focuses to 

a different location from that of

the ssDNA because of its 

different eletrophoretic mobility 

and a fluorescent band is 

observed. If the ssDNA is not

complementary to the PNA, a

focused fluorescent band is not 

observed because the neutral 

PNA is not focused by TGF. 

Two powerful features of TGF were

demonstrated in this work: 

• the ability to concentrate charged analytes 

• the ability to hold analytes stationary in a 

microfluidic channel for mixing and 

subsequent binding with a probe molecule 

by bulk flow of the probe through the 

analyte zone. 

Fluorescence micrographs of focused DNA and PNA/DNA in a microcapillary. In the 

experiment the ssDNA is first focused by TGF. PNA is then introduced into the channel by

bulk flow while the zone of concentrated ssDNA is held stationary by TGF. The ssDNA is 

labeled with a green fluorescent dye, while the PNA is labeled with an orange fluorescent 

dye. The top two fluorescence images are those obtained after focusing of the perfect 

ssDNA complement (initial) and then 15 min after introduction of the PNA (final). The bottom

two images are control experiments where ssDNA target that is non-complementary to the 

PNA is first focused (initial) and then the PNA is flowed for 27 minutes (final). Because the 

neutral PNA does not bind to the ssDNA, a focused orange spot is not observed in this

case. Each image is ≈ 2 mm long. In these experiments the ssDNA targets were

fluorescently labeled to confirm that the PNA/DNA duplexes are focused to a different 

location. In a real assay, it would not be necessary to label the ssDNA targets. 

109
Micellar Affinity Gradient Focusing 

D.J. Ross and K.M. Balss (836); W.N. Vreeland and P.B. Howell (839) 

Micellar affinity gradient focusing (MAGF) is a new method for the focusing and concentrating

hydrophobic and neutral analytes in microfluidic channels or capillaries. MAGF works by creating 

a gradient in the retention of the 

analyte into the micelles as illustrated 

in Fig. 1. On one side of the retention 

factor gradient, the analyte is strongly 

partitioned into and moves with the 

micellar phase. On the other side, the 

analyte is weakly partitioned into the

micellar phase so that it moves with 

the mobile phase (buffer). Using 

charged micelles and the combined 

application of an electric field and a pressure gradient, the micelles can be made to move from

the region of high retention to the region of low retention while the mobile phase moves from the 

region of low retention to the region of high retention. 

When this is done, the net velocity of the analyte will 

be positive on one side of the gradient and negative 

on the other side and zero at some point in the 

middle. Analyte will then move towards the zerovelocity point from both directions and be focused

and concentrated there. Different analytes, with 

different affinities for the micellar phase, will be focused at different points along the channel, so

that the method provides a focusing mode 

analog to micellar electrokinetic 

chromatography (MEKC) separation. 

Unlike previously described focusing

separation methods (such as isoelectric 

focusing (IEF), electric field gradient

focusing (EFGF), or temperature gradient

focusing (TGF)), MAGF provides for

separation of analytes based on

properties – such as hydrophobicity – that

are not related to the electrophoretic 

mobility. Consequently, it can be used to 

focus neutral and zwitterionic molecules 

that cannot be focused by previously described methods (Fig. 2). 

A new focusing method, micellar affinity 

gradient focusing (MAGF) was invented to meet 

the need for effective pre-concentration 

techniques in microfluidics. This method is the 

first electrokinetic focusing method that 

provides the ability to concentrate and separate 

analytes based upon properties other than 

electrophoretic mobility or isoelectric point.

Initial experiments indicate that MAGF 

can provide a simple method for 

focusing classes of analytes – such as 

neutral analytes – that cannot be

electrophoretically focused by other 

means. 

Fig. 1. Schematic of micellar affinity gradient

focusing in a microchannel or capillary. 

+V

micelles

high retention low retention

analyte

Fig. 2. Micellar gradient focusing and separation of two zwiterionic 

fluorescent dyes, rhodamine B (red) and rhodamine 110 (green). For 

scale, the image is 2 mm long. 

110
Microanalytical Technologies to Support NIH Measurement Programs 

L.E. Locascio (839); M. Gaitan and D. Reyes (812); N. Morgan and P. Smith (NIH/OD/ORS); 

T. Pohida (NIH/CIT); T. Phillips (NIH/OD/ORS); E. Perruccio (NIH/NEI) and P. Becerra 

(NIH/NEI) 

The purpose of this collaborative

effort is to fabricate microsystems 

based on microelectromechanical 

systems (MEMS) and microfluidics 

to support and improve the 

measurement capabilities of

medical researchers at the NIH 

laboratories.

Project 1. Human Papilloma Virus 

(HPV). The first project involves an 

epidemiological study of the

immune response to HPV, for which

the simultaneous isolation of

multiple proteins from microliter 

samples of cervical secretions is 

required. For this purpose, we are

developing a microfluidic system

capable of multi-analyte detection in 

a single small volume sample by performing multiple sequential heterogeneous immunoassays 

on chip. Microfluidic channels designed in a serpentine pattern are fabricated at NIST using 

silicon and glass substrates as shown in the figure.

Different antibodies are then covalently immobilized to

each separate arm of the chip so that the system is 

capable of measuring a number of analytes equal to the

number of arms. The channel device architecture has 

several advantages over existing array technology 

including the fact that the device is reusable, and the 

captured proteins can be extracted after measurement 

with their biological activity intact. We are designing the

system so that the basic methodology can be applied to 

many different applications relevant to clinical and

biological research at both institutions. 

We recently established collaborations with NIH for 

the purpose of enhancing medical research using 

microfluidics and microengineering. There are two 

ongoing projects with teams at NIH whose research 

involves: 

• the immune response to Herpes Papilloma Virus 

(HPV) 

• vision-related diseases involving defective 

neuronal differentiation or cell survival 

For the first project, we are developing chip-based 

microfluidic devices for use in multi-analyte 

immunoaffinity capture and detection of proteins 

related to HPV in cervical secretions. The second 

project involves patterned immobilization of

mammalian retinal cells using microfluidic channels 

to guide their growth and facilitate observation of 

their behavior under different conditions and 

treatments. 

Project 2. Visionrelated disease. There exist a number of techniques that 

have been used for patterning cells on surfaces. The

strategy often used to adhere single (mammalian) cells 

involves immobilization of extracellular proteins onto the

surface for further adhesion of the cells by interaction with 

these proteins. In this work, immobilization of rat retinal

epithelial cells is accomplished using micropatterned

polyelectrolyte multilayer (PEM)-coated surfaces. PEMs 

were deposited in discrete lines using a (poly)dimethylsiloxane (PDMS) microfluidic network on top of a flat 

PDMS slab. The layers were formed by sequentially

flowing the polyions throughout the microfluidic network.

Retinal cells, seeded on flat PEMs/PDMS surfaces 

Microfabricated silicon/glass device 

with 4 arms for antibody capture. 

Cervical sample 

Different 

antibody on 

each arm of 

device 

Waste 

Schematic diagram of device 

with 6 arms for measurement of 

6 analytes. 

111
adhered and grew on

the PEM areas 

preferentially as shown

in the figure at right. 

Cells were allowed to

grow for up to two 

weeks showing the 

pattern delineated by 

the PEMs. We believe 

this approach could

provide a useful and

simple tool to pattern

single cells or cell

networks in specific 

locations to monitor 

neural activity of retinal 

cells as they are 

exposed to different

stimuli.

0.88 cm

A) Overnight growth (~18hr) of retinal cells on 

poly(ethyleneimine). Cell growth is observed on the polycation 

surface areas (within the dotted black lines). B) Two weeks cells’ 

growth on poly(ethyleneimine). Cells are covering all the 

polycationic area. 

 B 

 A 

Development of Reference Methods and Reference Materials for Clinical Diagnostic 

Markers 

M.J. Welch, D.M. Bunk, M. Satterfield, L.T. Sniegoski, and S.S-C. Tai (839) 

NIST has a long-standing effort to

promote accuracy in health-related

measurements through development of 

reference methods and SRMs.

Certification measurements have now 

been completed for SRM 2921 Cardiac 

Troponin I, an important marker for heart

attacks. (See report titled “Development 

of SRM 2921, Human Cardiac Troponin 

Complex: A Primary Calibrator for Assays 

Used to Diagnose Heart Attacks.”) 

Method development has been completed

for triiodothyronine (T3) a marker for thyroid function, and participation has begun in an

international round-robin comparison for thyroid markers. (See report titled “Development of

Reference Methods and Participation in

International Round-Robins for 

Determination of Thyroid Markers.”) 

Work has begun on a new SRM for 

homocysteine (HCY) and tetrahydrofolic 

acid (FOL) in serum. This new material 

will have three levels: (high HCY, low 

FOL; normal HCY and FOL; and low 

HCY, high FOL). HCY is considered a 

risk factor for heart disease and other 

diseases associated with oxidative

damage, while FOL is a strong

antioxidant, which is known to reduce the 

risk of neural defects in fetuses and is 

NIST is committed to promoting accuracy and 

providing measurement traceability to the U.S. 

in vitro diagnostic (IVD) industry through 

development of reference methods and SRMs. 

To meet new regulatory requirements and help 

the US IVD industry compete globally, new 

reference methods for disease markers and 

metabolites are being developed here and in 

collaboration with other national metrology 

institutes. In addition, new SRMs are being 

developed for disease markers and important 

existing clinical SRMs are being renewed. 

Activities Completed or Underway 

• New methods SRMs in serum for 

• cardiac troponin I 

• homocysteine and folate 

• Methods development and participation in intercomparisons for thyroid hormones in serum 

• Collaborative reference method development for

C-reactive protein with NMIs in UK and Germany

• Renewal of existing clinical frozen serum SRMs 

for: 

• cholesterol and triglycerides 

• electrolytes 

• glucose 

112
believed to counteract the effects of homocysteine. The new material will be ready for

certification measurements in late 2003. 

Research has begun on development of a reference method for another risk factor for heart

disease, C-reactive protein (CRP). Modest increases in CRP have been linked to arteriosclerosis 

and the increased risk of heart attacks. NIST, working with scientists at the Laboratory of the 

Government Chemist (LGC) in the UK and the Physikalisch-Technische Bundesanstalt (PTB) in 

Germany, is planning a proteomics approach to isolating characteristic peptides from CRP for 

measurement by liquid chromatography/mass spectrometry. Measurements have begun for the 

certification of new lots of SRM 1951b Lipids in Fresh-frozen Human Serum, SRM 956b

Electrolytes in Frozen Human Serum, and SRM 965a Glucose in Frozen Human Serum, three of

the most used clinical SRMs. 

Development of SRM 2921, Human Cardiac Troponin Complex: A Primary Calibrator for 

Assays Used to Diagnose Heart Attacks 

D.M. Bunk and M.J. Welch (839) 

After evaluation of six different candidate

reference materials using two round-robin 

studies, a single reference material has 

been chosen and used to prepare SRM 

2921, the first reference material for cTnI 

measurements. More than 20 different

commercial cTnI assays, made by 12 

different manufacturers from the United 

States, Europe, and Japan were used to

evaluate the human cardiac troponin 

complex chosen for SRM 2921. SRM 2921 

will be used by assay manufacturers

developing new assays and for validating

calibration materials for their current 

assays. 

The clinical measurement of serum cardiac 

troponin I (cTnI) has become an important 

tool in the diagnosis of acute myocardial 

infarction and myocardial damage. 

Unfortunately, a ten-fold difference in 

measured concentrations between assays 

is common, but as much as a hundred-fold 

difference in response has also been 

observed. To address the need for 

standardization of cTnI assays, NIST, the 

American Association for Clinical 

Chemistry (AACC), and the International 

Federation for Clinical Chemistry (IFCC) 

have worked together to develop SRM 2921, 

human cardiac troponin complex. 

The certification of SRM 2921 was accomplished through a 

combination of analytical methods, both qualitative and

quantitative. The values of the concentration of each of the

three troponin subunits, including cTnI, were determined using 

amino acid analysis coupled with a chromatographic 

separation. The structure of the cTnI protein was evaluated

using mass spectrometry coupled with chromatography to

determine the extent of post-translational modifications such as

phosphorylation and degradation of the protein chain. 

Data from the second 

round-robin 

demonstrated that the 

use of one common 

reference material could reduce interassay variability to

10-15 %, a substantial improvement compared to the 130

% variability observed in the first round-robin. A third 

round-robin study will be carried out to determine fully the ability of SRM 2921 to harmonize the 

cTnI assay results from patient samples and to establish its commutability. Future work will also

include the development of a reference method for cTnI in serum. 

In a recent issue of Clinical 

Laboratory News, the selection of 

a material to be used as a primary

cTnI standard was touted as an 

“important milestone” in the 

standardization of clinical cTnI 

assays. 

113
Development of Reference Methods and Participation in International Round-Robins for 

the Determination of Thyroid Markers 

S. S-C. Tai, D.M. Bunk, and M.J. Welch (839) 

A new reference method based upon 

isotope dilution combined with LC/MS/MS 

has been developed for serum T3, a 

marker for thyroid function. Development 

will begin in FY 04 for a human serumbased SRM for thyroid markers, T4 and 

T3, and other important hormones 

including cortisol, estradiol, progesterone,

and testosterone. 

The major challenges of serum T3

measurement are the low concentration of

T3 normally found in serum, and the 

degradation of serum T4 into T3 during sample preparation. Because the concentration of serum 

T4 is approximately 100-fold higher than that of T3, even slight degradation of T4 into T3 can 

have a strong influence on the measured serum T3 values. The T4 degradation in this reference 

method was minimized to a small percentage, and can be corrected for in the T3 measurement 

process. This method was critically evaluated and found to produce results of high precision and

accuracy for this low level hormone. This LC/MS/MS method for T3 along with LC/MS/MS method 

for T4 developed at NIST was used in international round-robin comparisons with excellent

agreement with laboratories in Europe.

In the past year, NIST has completed 

development of a reference method for the 

determination of serum total triiodothyronine 

(T3), a marker for thyroid function. This 

method is based upon isotope dilution 

combined with liquid chromatography and 

tandem mass spectrometry (LC/MS/MS). NIST

has also participated in international roundrobin comparisons of reference methods for 

the determination of thyroid markers, total 

thyroxine (T4) and total T3. 

LC/MS/MS ion chromatogram for serum

T3 marker 

The reference methods base on 

isotope dilution LC/MS/MS developed 

here and the SRM that will be used to 

certify these methods will provide 

critical traceability to the in vitro 

diagnostics industry and will help 

improve the reliability of routine 

clinical measurements. 

Improved Methodology for Determination of Arsenic in Body Fluids 

E.A. Mackey and R. Zeisler (839) 

The current INAA detection limit for As in

typical body fluids is generally between 1 

ng g-1 and 10 ng g-1. The Na, P, and Br in

the sample, however, elevate the

baseline level of radiation next to, and

under, the As peak of interest. Therefore, 

the wide As detection limit range

depends upon the amount of background 

produced by matrix elements such as Br, 

P, and Na. 

To quantify the lowest levels of arsenic 

typically found in body fluids, detection limits 

for instrumental neutron activation analysis 

(INAA) must be reduced by about an order of 

magnitude. In this work, improvements in the 

INAA detection limits for As in biological 

samples were investigated using improved 

detection systems and simple radiochemical 

procedures. 

114
Two approaches for decreasing As detection limits were investigated. The first approach involved

the use of a dissolution procedure to volatilize Br. Samples were digested with nitric acid and 

water and heated to near dryness. This was followed with heating with additional nitric acid and 

perchloric acids, again until near dryness. The digested material was diluted and counted using a 

conventional γ-ray spectrometry system consisting of a germanium detector and associated

electronics. The γ-ray spectra from these digests were compared with spectra from the intact

irradiated samples, acquired prior to digestion.

Results indicated that the Br-to-As ratio was 

reduced by a factor of two using this approach.

However, this reduction is not sufficient to permit 

quantification of As at the low levels present in

materials such as human urine supernatant (SRM 

2670a). 

The second approach involved use of electronics 

capable of processing high count-rates without degraded resolution so that counting could be

started earlier in the decay. The combination of a "zero dead-time" spectrometer with the latest

gamma-ray spectrometry software has permitted this technical improvement. The As-to-Br ratio in 

spectra acquired using this system was up to a factor of 4 higher than obtained using a 

conventional spectrometry system and peaks were well resolved. Using this system the limit of 

quantitation for a 12 h count of human urine supernatant is about 1 ng g-1. 

State-of-the-art γ-ray spectrometry as well 

as simple radiochemistry can improve

INAA detection limits for As in human 

body fluids. This work demonstrates that 

the use of more advanced electronic 

signal-processing systems can improve 

the limit of quantitation for As in 

biological fluids by a factor 4 or more. 

A simple volatilization procedure did not yield sufficient improvement to detect levels of As 

present in fractions of biological fluids. Use of a more sophisticated detection system was 

successful but required longer counting times, thereby limiting throughput. Additional work will 

involve investigating the utility of simple radiochemical separation procedures in conjunction with

the improved detection system. 

Reference Methods and SRMs for Toxic Species in Body Fluids 

S.E. Long, R.D. Vocke, L.L. Yu, K.E. Murphy, T.A. Butler, and E.A. Mackey (839) 

We have developed new analytical 

methods for the determination of trace 

and ultra-trace levels of toxic elements 

and cyanide in blood and urine. A 

species-specific method has been 

developed to accurately determine levels 

of inorganic mercury in whole blood. The 

method is based on selective reduction of

the inorganic mercury component, which

is combined with an ultra-sensitive

isotope dilution vapor-generation ICP-MS 

measurement. The organic mercury 

present in the sample is not detected.

The method has been applied to the 

determination of inorganic mercury in the 

elevated level of SRM 966 (Toxic Metals 

in Bovine Blood). This has been used to

upgrade the SRM certificate value for inorganic mercury from a reference value to a certified

value, which should be a valuable enhancement for the user community. 

Clinical assessment of the impact of toxic 

species on human health depends upon the 

quality of chemical measurements and the 

availability of suitable reference materials. 

Toxic species that are currently receiving 

scrutiny in the most common clinical matrices, 

blood and urine, present considerable 

measurement challenges since concentration 

levels are often close to the limit of 

quantitation by traditional methods. To 

address these challenges, we are developing 

new analytical methods with improved 

sensitivity and capability for toxic elements 

and cyanide in blood and urine. The methods 

have been used to augment the range of 

reference materials available from NIST. 

115
Certification of a replacement freeze-dried urine reference material (SRM 2670a, Toxic Elements 

in Urine) has been completed, mostly using a measurement approach based on isotope dilution

combined with the high sensitivity of sector field inductively coupled plasma - mass spectrometry.

The new material represents a considerable technical 

advancement compared with its predecessor, SRM 

2670, having ten elements certified at the natural 

background level and fourteen elements certified at the 

elevated (spiked) level. Many of these certification

measurements were made at the ultra-trace 

concentration level, which were possible as a result of

the availability of new high-quality cleanroom facilities. 

We have also developed an improved method for the

determination of cyanide in whole blood based on headspace gas chromatography with mass 

spectrometric detection (GC/MS), and employing K13C15N as the internal standard. Cyanide (CN) 

is a very fast acting poison, which is rapidly absorbed in the blood and inhibits the cellular

utilization of oxygen. Death can result from a blood CN concentration as little as 2.5 μg/mL. 

Despite its known hazard, CN is extensively 

employed in a number of chemical processes. Its 

presence in tobacco smoke and certain foods 

represent important health issues. The 

determination of cyanide is analytically 

challenging, and because it is unstable bloodbased standards are not commercially available.

The new analytical method is being used to 

provide value assignment and stability testing

data for a pool of baseline and cyanide-spiked 

blood samples produced by the Emergency 

Response and Air Toxicants Branch of the 

Centers for Disease Control and Prevention

(CDC). 

A new instrument system featuring 

a combined liquid chromatography

– inductively coupled plasma mass 

spectrometry system (LC-ICPMS)

with a dynamic reaction cell for 

reducing spectral interferences has 

been installed, and will be used to 

investigate the role of toxic species 

in human nutrition and disease. 

LC-ICPMS with dynamic reaction cell 

Radiochemical Method for Benchmarking Silicon Measurements Made During Clinical 

Investigations 

R. Zeisler and R.L. Paul (839) 

In recent years, the role of trace 

concentrations of silicon in biological 

systems, namely in the human body, has 

become of increasing clinical concern and 

research interest. Association of elevated 

silicon levels in blood components and

various tissues in patients with chronic renal 

failure and in women with silicon breast 

prosthesis was reported, as well as colocalization of silicon with aluminum in senile

plaques in the brain tissue of patients with 

Alzheimer’s disease. 

The analytical difficulties associated with the

determination of Si at low levels in biological materials were documented in a recent

The role of silicon in diseases and 

disorders has yet to be fully elucidated. 

This is mainly due to analytical difficulties 

associated with the low-level determination 

of silicon in biological materials, such as 

high and variable blank values or analyte 

losses. This work demonstrates that the 

use of a radiochemical neutron activation 

analysis (RNAA) procedure, involving the 

NIST reactor, can provide detection limits 

for Si in biological materials that are fit for 

value assignment of Si in benchmark 

SRMs. 

116
intercomparison on the element determination in

pork liver and pork filet samples. Used in this 

study were various modes of electrothermal 

atomic absorption spectrometry, inductively 

coupled plasma optical emission spectrometry, 

and direct wavelength dispersive X-ray 

fluorescence analysis. Severe discrepancies were found among results of these techniques, 

especially at the few μg g-1 level. In order to be able to improve the performance of routinely 

applicable analytical techniques, there is a need to develop biological SRMs with certified values

of silicon. The development of a new radiochemical neutron activation analysis (RNAA) method

is a critical step towards the development of appropriate benchmark SRMs. 

It was concluded that the challenging task 

of accurate low-level determination of Si in 

biological materials can be accomplished 

with the employment of the inherently

suitable properties of RNAA. 

The new RNAA method, utilizing the 30Si(n, γ)

31Si nuclear reaction with thermal neutrons, was 

developed for low-level determination of Si in biological materials. The radiochemical separation

consists of an alkaline-oxidative decomposition followed by distillation of SiF4. Nuclear

interferences, namely that of the 31P(n,p)31Si with fast neutrons, have been examined and found 

negligible only when irradiation is carried out in an extremely well-thermalized neutron spectrum,

such as available at the NIST reactor. The RNAA procedure yields excellent radiochemical purity 

of the separated fractions, which allows the measurement of the β-

 activity of the 31Si by liquid

scintillation counting. Interference-free detection limits achieved were 150 μg g-1 for irradiation of 

200-mg samples in the NIST reactor RT-4 channel for 30 minutes, and 0.5 μg g-1 for irradiation of 

500-mg samples in the RT-5 channel for 10 h. Initial values have been established for SRM 1549

and SRM 1577b. Future plans call for implementing and refining the RNAA method for assigning

values of the Si content in clinically relevant SRMs such as SRM 1598a.

Standards for Fluorescence Microarray Analyses 

G.W. Kramer (839); A.G. Gaigalas (831); P.C. DeRose (839) 

DNA microarrays, also known as DNA or 

gene chips, have become important tools for 

gene expression analyses and are poised to 

revolutionize clinical diagnostics and enable 

personalized medical care—where treatment 

can be individually tailored to a specific 

person through genetics-based diagnoses. 

The related protein microarrays show great 

promise for pharmaceutical drug discovery 

research as well as clinical diagnostic tools.

For microarrays, single-stranded genetic or 

protein material (probes) is bound in an array 

to a surface the size of a standard

microscope slide permitting tens of thousands of molecular reactions to be tracked in parallel 

when an analyte solution is washed over the

array. To accommodate this many sites in such a

small area, the individual spots of genetic 

material or protein must be very small, commonly 

on the order of 10 μm to 100 μm in diameter.

Target DNA or protein in the analyte solution are

labeled with a fluorescent dye and allowed to 

interact with the probes bound on the surface of

the array. A favorable interaction leads to 

U.S. manufacturers of microarray scanners 

have asked NIST to develop measurement 

standards for fluorescent microarray 

analyses. Specifications for a series of 

physical artifacts have been developed 

during several NIST workshops and 

monthly teleconferences. Work continues 

in finding suitable fluorescent materials, 

identifying appropriate fabrication 

methods, and creating instrumentation for 

certifying the fluorescence of such 

standards.

If artifact standards for microarray assays

can be created and certified, then assay

quantitation can be improved, results will 

be intercomparable, and the measurements 

can be made traceable ultimately back to 

the SI. The purpose of this project is to 

develop such standards in collaboration 

with the manufacturers of microarray

readers

117
fluorescence signal from the appropriate spots. A device called an array scanner or reader 

detects which spots in the array fluoresce and how much. 

Today, there is no consensus method for microarray assays; there are multiple technologies for 

fabricating microarrays ranging from nano-pipetting to pin-printing to photolithography, and there

are several types of scanners. The method variations and uncertainties are so great currently that

the biological information obtained from microarray assays is often a function of the method and

equipment used, and comparisons of results generated by differing technologies is not possible. 

Prompted by a request from the scanner industry for standards development by NIST, a

workshop on fluorescence standards for microarray assays co-sponsored by Agilent

Technologies and NIST’s Biotechnology and Analytical Chemistry Divisions was held on 

December 10, 2002. Attended by representatives from the major array reader manufacturers, its 

purpose was to develop technical specifications for fluorescence intensity, uniformity, and 

detection limit standards for the

calibrating and validating microarray 

readers. Several parameters such as the 

excitation and emission wavelengths of

the fluorescent tags seemed to be

common across the industry; however,

there were diverging views on other 

specifications such as spot size and the

form factor of the array. 

Following the initial technical workshop, 

the participants worked out many issues 

concerning the types of standards,

intensity levels, form factors, etc. in 

teleconferences. It was agreed that

unpatterned artifacts for each of two 

colors (similar to the dyes Cy-3 and Cy-5

that are commonly used now) would be developed. One set

of artifacts with fluorescence intensities in the mid-to-high 

range would serve as uniformity/homogeneity standards and to measure signal-to-noise ratios for 

bright features, while a second set of materials would be developed with low fluorescence levels

to serve as detection limit standards and to measure signal-to-noise ratios for dim features. To

avoid having to accommodate separate sets of

standards to serve the two predominant form 

factors (25 mm by 75 mm microscope slide and ≈

15 mm square), it was agreed that the artifact

would be made oversize with two active areas that 

could be separated by breaking the glass along a

scribed line. 

Example of microarray readout 

A second technical workshop was held at NIST on

May 14, 2003 to come to agreement on many of

the physical factors and to begin the search for 

appropriate fluorescent materials and application

processes to fabricate the standards. Through monthly teleconferences, the group continues to

refine parameters and to search for suitable materials. 

NIST sponsored workshops: 

• December 10, 2002: a workshop on 

fluorescence standards for microarray

assays co-sponsored by Agilent 

Technologies and CSTL.

• May 14, 2003: workshop to begin the 

search for and fabrication of 

appropriate fluorescent materials for

standards.

118
9. Industrial and Analytical Instruments and Services

Instrument manufacturers depend on NIST/CSTL for physical and

chemical standards, as well as NIST databases, for instrument 

calibration to provide traceability to national standards to the end 

users of their products and instruments. CSTL work supports both

mature analytical applications, as well as the newest technologies 

such as high-throughput screening, and cutting edge research for 

next-generation semiconductor manufacturing. 

Calibration of a Gamma-ray Detector for a Mission to the Planet Mercury 

R.L. Paul, G.P. Lamaze, and R.M. Lindstrom (839); E.A. Rhodes (Johns Hopkins Univ.)

The NASA MESSENGER spacecraft, designed and built by scientists and 

engineers at the Johns Hopkins University Applied Physics Laboratory, will

become the first mission to orbit the planet Mercury. Once in orbit, a

gamma-ray spectrometer aboard MESSENGER will map the gamma-ray 

emission spectra over the planet resulting from capture of cosmic ray

induced neutrons by nuclei of elements in the planet’s crust. Analysis of 

these spectra will allow determination of the composition of Mercury’s crust, 

which will shed light on the evolution of this planet and our solar system. In

order to get an accurate assay map, the gamma-ray detector must be calibrated for efficiency vs. 

energy over the spatial orientation angles associated with

the orbits around the planet. This calibration is usually 

performed using calibrated radioisotope sources.

However, radioisotope sources have weak or nonexistent 

gamma-ray emission at energies above 3 MeV, while the

gamma-ray energies of importance for planetary assay 

extend up to energies of around 9 MeV. An additional 

problem facing JHU scientists was that the germanium 

detector must be cooled by a small cryocooler that has an estimated lifetime not much longer 

than the mission. Therefore, the calibration measurement could not be allowed to consume much

time. Because prompt gamma-rays span the energies 

needed for the calibration, the only viable method for 

obtaining a timely and accurate calibration of this 

detector was to use gamma-rays produced by neutron 

capture of a target in a high intensity neutron beam.

Measurements were made using the unique beam 

facilities of the NIST Center for Neutron Research. 

Targets made of NaCl and Cr, irradiated at the NG0

beam port, provided the necessary gamma rays for 

calibration of the MESSENGER detector, which was 

rotated to the angular orientations required for the 

space flight. In order to obtain an absolute efficiency 

calibration, radioisotope sources of 56Co, 228Th, and

226Ra were calibrated at NIST and were measured in 

the same configurations as the targets. The 

spectrometer has been loaded aboard the spacecraft.

This calibration procedure will allow MESSENGER to 

yield a relatively accurate compositional map of 

Mercury, and will likely set a new standard for spaceflight gamma-ray spectrometers.

CSTL scientists developed the 

calibration procedure for the 

Messenger Spacecraft that 

meets the time and energy 

parameters required by the 

mission engineers.

100 1000 10000

Radioisotope sources

Prompt gamma sources

emitted gamma ray)

Energy (keV)

An efficiency calibration determined 

using radioisotope sources of 56Co, 

228Th, and 226Ra measured in the 

same configurations as the targets. 

119
Standards for Raman Spectroscopy 

W.S. Hurst and J.E. Maslar (836); S.J. Choquette (839); E.S. Etz (837) 

120

Raman shift (cm -1 )

4000 Intensity (arb. u.)

3000 2000 1000 0

SRM 

2242

532 nm

Raman spectroscopy is now finding its place in the 

industrial environment for process measurements and 

quality control. NIST standards will promote the 

acceptance of Raman spectroscopy as a tool for

process control in the chemical and pharmaceutical

industries and will provide a means for instrument 

qualification as required by regulatory agencies such

as the FDA. The project critically evaluates existing approaches and develops new methods and

associated standards that will provide for 

calibration of the intensity of Raman spectral 

data. Intensity calibration is needed to make

process-control Raman measurements 

“instrument-independent,” to enable analysis of 

spectral data for unknown mixtures, and to

quantify instrument performance and stability 

as needed by U.S. industries. CSTL scientists

are developing a series of fluorescent glasses 

at will become available as Standard

eference Materials (SRMs) and will provide

strument calibration. SRM 2241, a glass 

suitable for use with laser excitation at 785 nm, 

was made available last year. A new fluorescent glass with stable properties suitable for use with

laser excitation at 532 nm, SRM 2242, will be available in early 2004 and will supply the glass 

artifact along with a curve expressing its relative irradiance as a function of the Raman Shift in 

wavenumbers. SRM 2243, for 488 nm and 514 nm laser excitation, is under development and 

expected to be available in late 2004. It has been 

examined by members of the ASTM E13.08 for 

user advice on the magnitude of the intensity 

response, in order to establish the final 

fluorophore concentration. While the final 

fluorophore concentration has been determined, 

additional NIST work is needed to minimize laserbleaching effects. In response to requests by 

members of ASTM E13.08 for NIST to include in 

its suite of materials for intensity calibration one for use at 633 nm, NIST will develop and 

complete a SRM for 633 nm in the next two years. 

CSTL scientists are providing 

chemical and pharmaceutical 

industries with an inexpensive 

and easily implemented means 

for calibration of the Raman 

spectral intensity. 

th

R

in

• SRM 2242, a manganese-oxide bearing 

glass suitable for use with 532 nm laser 

excitation has been developed and will

be available in early 2004. 

• This will be followed by SRM 2243 for 

488 nm and 514 nm expanding the series 

and enabling this technique’s use to be 

expanded to the pharmaceutical, 

chemical, and forensics communities.

Fluorescence Standards Suite for Spectral Correction of Fluorometers 

P.C. DeRose, D.H. Blackburn, and G.W. Kramer (839) 

Luminescence measurements have become the detection methods of choice for new clinical and

biochemical assays, and related high-throughput 

techniques, due to their extraordinary selectivity and 

sensitivity. These new analytical methods are becoming 

increasingly quantitative, requiring standards to calibrate 

the luminescence measuring instruments that they 

utilize and aid in the validation of the methods. Ideally, 

users would like to employ the same organic dye probes 

used for analyte detection as standards for fluorescence

intensity and spectral correction. Unfortunately, organic 

CSTL researchers are working 

to prepare, characterize and 

certify a set of fluorescent glass 

SRMs that will enable spectral 

emission to be calibrated on 

fluorometers in the near UV and 

visible regions of the spectrum.
dyes photobleach quickly, do not have long shelf lives in solution, have environment dependent

fluorescence and are expensive to produce at high purity. After studying the characteristics of the 

different types of fluorescent materials, we found metal oxide doped (MOD) glasses to be the

best choice for use as fluorescence standards for spectral correction. These glasses are

photostable, robust, relatively inexpensive and can be made to suit most detection formats. Our

research-grade fluorometer is being used to

characterize the spectral characteristics of a series 

of MOD glasses to determine which will have the 

correct spectral shape and intensity for appropriate

standards. A UV light chamber and a laser are also 

being used to irradiate the candidate glasses over

a timed period. The fluorescence intensity of each 

glass is measured before and after irradiation to determine its rate of photobleaching.

The certified U3O8 and MnO2 glasses can 

be used in combination with SRM 936, a 

quinine sulfate dihydrate; a blue spectral 

emissivity standard that is the only

standard presently sold for spectral 

correction of fluorescence

Nanowires on Microhotplates 

R.E. Cavicchi and C. Montgomery (836); B. Nikoobakht and S. Stranick (837) 

Sensing devices based on nanowires and 

nanorods hold great promise for applications in 

chemical sensing. Small size implies that a greater 

fraction of atoms of these nanomaterials lie near

the surface, where interaction with the 

environment can provide measureable changes in 

the electrical and optical properties. Of particular 

interest is the concept of a “nanolaser,” where the 

dimension of the optical element is small 

compared to the wavelength of light. Lasing has 

only been observed in nanorods by optical pumping for a few systems, and only once by the 

much more practical electrical-driven pumping for a device 

geometry that is suitable only for demonstrations. There has 

been much excitement recently over the development of growth

methods for nanowires for optical materials. Based on 

techniques used to prepare carbon nanotubes, these methods 

involve the use of finely dispersed metal catalysts that promote 

the growth of crystals in one dimension from a vapor that 

passes over them. This work represents the first formation of

nanowires on the electrical contact of a micromachined

structure. Vapor phase transport of ZnO powder in argon was 

used to deposit ZnO nanowires on a microhotplate prepared 

with an ultrathin film of gold metal islands. Growth temperature

was 1000 °C. Nanowires of average diameter 50 nm were

observed on the tungsten-based contact pads of the

microhotplate. The growth was selective to the contact

pads. This shows the compatibility of nanowire growth 

with MEMS (microelectromechanical systems) devices 

and establishes a mode for producing electrical contacts.

CSTL research represents the first 

formation of nanowires on the 

electrical contact of a micromachined 

structure. Vapor phase transport of 

ZnO powder in argon was used to 

deposit ZnO nanowires on a 

microhotplate prepared with an 

ultrathin film of gold metal islands. 

Growth of ZnO nanowires on 

microhotplate. 

ZnO nanowire structures can be 

grown selectively on the contact 

pads of a microhotplate or similar 

thermally-agile MEMS structure. 

121
Hot Electron Chemicurrents Produced in Chemical Processes at Surfaces: A Theoretical 

Model 

J.W. Gadzuk (837)

Understanding the mechanisms of energy dissipation in

surface reaction dynamics is necessary for useful predictive 

modeling of processes such as those that occur in vapor 

phase deposition and fabrication processes on surfaces.

Newly discovered phenomena in which small but measurable 

currents are produced when molecules interact with metal 

surfaces has provided the empirical basis for a new generation 

of detectors in which the chemical event itself generates the hot electrons that are detected. The

purpose of this theoretical work is to provide the foundation for some aspects of the underlying 

physics that are operative in these hot electron-based devices. 

CSTL researchers have 

discovered small but 

detectible currents 

produced when molecules 

interact with metal 

surfaces. 

The crucial role of substrate electronic excitations in limiting

the reaction rate of chemically selective atomic and molecular 

surface processes such as sticking/adsorption/dissociation/

inelastic scattering has long been debated. Recent

experiments carried out at the University of California at 

Santa Barbara have detected hot electrons produced when

various gases were adsorbed on a thin metal film that formed 

a Schottky barrier with a Si substrate upon which the film was

deposited, as illustrated in the figure to the left. A theoretical

model for the electronically non-adiabatic effects has been 

developed that accounts for the observed initial hot electron 

production and subsequent detection as a “chemicurrent”,

roughly ≈ (0.001 to 0.01) electrons per incident stronglyinteracting adsorbate. This is based upon analogies with

electron-hole pair shakeup processes that occur in the

dynamic response of metallic systems to transient localized perturbations first addressed within 

the context of core level spectroscopies of solids. 

Fig. 1. Chemically induced 

hot-electron detector. 

The theory of chemicurrents includes a twofold thrust 

describing: i) the hot electron production, symbolized by 

the starburst in Fig. 1; and ii) the solid-state detector 

characteristics of the metal film and Schottky barrier also

shown in Fig.1. A flux of reactants characterized by some

incident energy ∇η and an interaction strength parameter

α (simply related to the charge transfer between the 

reactant and the surface) creates the same distribution of 

excited electrons as occurs in core state spectroscopies 

in which the core electron is excited to a kinetic energy 

∇η. The theoretical predictions for the hot electron energy 

distribution as a function of scaled energy which follow

from previously developed and confirmed spectroscopy 

theory, are shown in Fig. 2 (top), for events characterized

by charge transfer in the not-unreasonable range ≈ 0.25 

to 1.0 electrons (0.05<α<0.25). Using these excited state 

distributions in conjunction 

with theory for the thin film 

detector, the predicted

chemicurrent or electron 

yield per incident

Fig. 2. (Top) Hot electron 

energy distribution. 

(Bottom) Chemicurrent yield 

vs. chemisorption energy for 

labeled incident energies. 

This technology “...may

usher in a new generation

of chemical detectors.” 

(NSF) 

122
adsorbate is shown in Fig. 2 (bottom) as a function of (essentially) the chemisorption energy, for

various incident energies. The predicted yields of about 0.01 electrons/adsorbate are in excellent

agreement with the observed yields for appropriate adsorbates such as O, H, and NO2. 

This theoretical work highlights some of the underlying system parameters that affect hot electron 

he NIST Mass Spectral Library - Improving the Evaluation

.A. Mirokhin (Contractor) 

ince the latest version of the NIST Mass Spectral Database 

The new version of the MS Interpreter takes into account experimental

t

co

ex

data currently in the Da

he GC/MS instrument is one of the most widely used tools 

e number of upgrades to the new 

production as a result of a gas/surface reaction, and results are indeed consistent with the salient

features of the experimental work. 

T

S.E. Stein, A.I. Mikaia, C.L. Clifton, J.K. Klassen (838); and Y

S CSTL researchers work to 

(NIST02) release, the focus has been on tools and methods 

to improve the evaluation of the data in preparation for future

releases. Ion-thermochemistry data are being used to rank

probability of ion occurrence. Work is continuing on both the 

development of standards for Gas Chromatograph/Mass

Spectral (GC/MS) libraries. As always, we continue to work

closely with the instrument manufacturers and the user

community to define the needs for the MS Database. 

provide mass spectrometris

with a comprehensive, reliable 

library of mass spectral 

‘fingerprints’ to assist the

of compound identification 

using the method of GC/MS.

and estimated heats of formation for ions. In addition, it finds multiple 

paths for ion decomposition. As a result of these improvements, a 

far larger fraction of the peaks in a typical mass spectrum are 

being identified. While the Interpreter is being developed as a tool 

for evaluation of NIST MS data, it is also a useful tool for quickly 

understanding a new mass spectrum. A candidate molecule can

be drawn and the Interpreter will take the structure and compare it 

o the data from the unknown. While such analysis does not

nstitute a proof of the structure, it can reduce the effort of the 

perimenter considerably. In addition to tools to better evaluate the

tabase, some changes in the direction of new data acquired have been

made. The NIST MS Database has had fewer of the important derivative spectra than it should.

These spectra are of compounds that are not easy to analyze by conventional GC/MS. Therefore, 

a derivative is made, which is easier to analyze. The largest groups of these derivatives are

methyl esters, which are well represented in the database, and trimethylsilyl esters, which are

underrepresented in the database. The emphasis on the addition of these derivatives as well as 

the continuing addition of spectra of newly discovered drugs, drug metabolites, and natural

products will insure that the NIST MS Database meets the needs of the GC/MS community.

T

for analysis of organic compounds. The efforts at NIST to

provide both the best data and tested methods for accessing 

that data have led to a wide acceptance of the NIST MS 

Database and the NIST search software as the best

combination of tools to use for MS analysis. With over 2,500 

new libraries installed on instruments each year and a larg

version of the Database, the higher quality and increased coverage of the new version will 

continue to improve search results in a large number of laboratories. 

ts

 task 

Progress is being made 

towards the next release

will include new evaluation 

tools and an ongoing 

examination of the stru

and naming of compounds. 

that

cture 

123
The NIST Retention Index Database 

S.E. Stein, P. Linstrom, and W.G. Mallard (838)

One of the major problems in the use of gas 

chromatography combined with mass spectrometry in

compound identification is the similarity in mass spectra

of homologous series. This problem has traditionally 

been solved by the use of retention index data (a 

measure of the time a compound elutes from the 

chromatographic column), but data and prediction for 

retention indices have not been widely available. The collection and evaluation of the retention 

index data will provide a new tool for the identification of organic compounds. The initial release

of the database (expected early in FY 2004) will contain data for 9000 chemical compounds along 

with complete literature references. During 2003 the first release of the database was prepared.

Researchers are checking the release to confirm that all structures and references are present. 

In addition, the first stage of the collection of data for the second release has begun. Data for 

over 14,000 compounds are expected to be present with the second release. In addition, a

scheme for using group additivity methods for predicting retention index data has been

developed. The prediction scheme is used to find outliers in the data and allow for data checking

with minimal resources. In addition, the prediction scheme will be available as a part of the

WebBook. 

The use of retention data 

provides a far greater degree of 

confidence in the identification 

than mass spectra or retention 

index alone would be capable of 

providing. 

The broad question of confidence in identification using 

modern analytical methods remains important. The use of

retention data - which is an orthogonal set of data to mass

spectral data - provides a far greater degree of confidence in

the identification than either mass spectra or retention index

alone would be capable of providing. This additional 

confidence is used in NIST deconvolution software to 

determine a match factor that takes into account all that we know about an identification.

Retention data has been used in certain very narrow areas - particularly in natural products or 

petroleum analysis, but the lack of high quality data sets has made it difficult to use retention data

for a broad scope of problems. 

There is a very substantial 

body of data that has been

identified and is in the process 

of being digitized and analyzed 

for inclusion in the next 

version. 

Automatic Mass-Spectral Deconvolution and Identification Software (AMDIS) 

W.G. Mallard, S.E. Stein (838); and O.V. Toropov (Contractor) 

The Automatic Mass-spectral Deconvolution and Identification Software (AMDIS) was originally 

designed to detect chemical weapons in violation of the Chemical Weapons Convention while at

the same time not reveal confidential information that was not relevant to the Convention. The 

same fundamental algorithms would clearly be of use in 

automating the analysis of data taken in gas

chromatography/mass spectrometry (GC/MS) in an industrial 

environment. In these cases the role of AMDIS is to provide

faster, unbiased, and more statistically testable results - thus 

allowing higher quality analysis. The major efforts over the last

year have been focused on assisting the 

instrument companies in using AMDIS in 

conjunction with their instrument software. 

CSTL scientists work to 

extend the utility of the 

AMDIS software and 

insure that it is available 

to the widest range of 

users.

During 2003 presentations were made to several industrial users at their sites 

with an emphasis on using AMDIS in solving their problems. In addition, 

detailed presentations were made to two major instrument manufactures to aid

124
in their integration of AMDIS into their instruments. On-going activity includes changes in library 

handling to allow for more records in the libraries and new modes of searching. In all cases these

changes have come about from direct requests from the users or the instrument companies. 

AMDIS offers a tool for finding, with high confidence, components 

in complex mixtures that would otherwise be impossible. In 

addition, AMDIS can significantly increase the productivity of 

analysts in the laboratory by doing the first pass analysis in 

minutes without direct intervention, rather than hours - with full 

involvement of the analyst. AMDIS can also aid in providing 

statistically valid confidence measures for analysis. There is a growing awareness in the GC/MS 

community of the utility of AMDIS. 

AMDIS provides the first 

broadly available method

for automating GC/MS 

analysis. 

Determination of Hydrogen in High-Purity Niobium for Particle Accelerators 

R.L. Paul (839), H.H. Chen-Mayer (PL), and G.R. Myneni (Jefferson Laboratory) 

Niobium RF superconducting cavities are fast becoming the basic 

accelerator structures of a new breed of particle accelerators. It is 

suspected that the presence of interstitial elements in the niobium

used to build these cavities may result in degradation of mechanical,

thermal, and electrical properties. Prior to use, the high-purity

niobium is chemically polished (treated with acid) to remove surface

imperfections that could degrade the superconducting properties.

This chemical polishing is believed to introduce trace hydrogen, while

vacuum heating may be used to remove it. Hydrogen measurements 

were made at the Center for Neutron Research using two methods: 

cold neutron prompt gamma-ray activation analysis (PGAA), which

measures gamma-rays emitted

upon neutron capture; and neutron

incoherent scattering (NIS), which measures neutrons 

scattered from the sample. The latter technique takes 

advantage of the anomalously high neutron scattering cross 

section for hydrogen. The samples were then acid treated with

applied voltage for (0.5 to 5) min, and again analyzed. The 

measurements indicated no change in H concentration after 

the acid treatment. To see if more rigorous treatment could 

induce changes, two samples were again vacuum degassed at

800 °C for 8 h, and one sample was again acid treated for 

5 min. Both techniques measured a significant decrease for the degassed sample, and a

significant increase for the acid treated sample, indicating that both processes can affect the H

content of the niobium. One possible explanation for the observed results is that heat treatment

at 800 °C causes removal of strongly bound hydrogen, thereby activating sites where hydrogen 

can be picked up by the niobium upon subsequent acid treatment. 

CSTL researchers have 

used prompt gamma-ray 

activation analysis (PGAA) 

and neutron incoherent 

scattering to study the 

effects of these two 

processes on the 

hydrogen content of the 

niobium. 

The results of this and future research will lead to newer and better ways to manufacture and

dehydrogenate high purity niobium. The ultimate benefit will be a significant decrease in the cost

of building accelerators to study fundamental physics. Since many more accelerators are 

planned for the future, the studies we are doing on niobium have a wide impact on the entire 

landscape of accelerator related science and technology.

125
Characterization of the New Thermal Neutron Prompt Gamma Ray Activation Analysis

Instrument 

E.A. Mackey, R.M. Lindstrom, H.H. Chen Mayer, G.P. Lamaze (839); D.L. Anderson (USDA);

P.J. Liposky (NCNR) 

A new thermal neutron prompt γ-ray activation analysis 

(PGAA) instrument was built at the NIST Center for 

Neutron Research. It replaced the original PGAA 

instrument that was built in the late 1970's. This new 

instrument provides greater sensitivities, better detection

limits, and much lower background radiation. The new 

instrument is also easier and safer to assemble for use. 

A sapphire filter was placed in 

the neutron beam shutter assembly to reduce the fast neutron and low 

energy γ-ray components of the beam. The fast neutron component of 

the beam was reduced by a factor of five and the low-energy

background γ-radiation by factors of 5 to 10. A new external beam 

tube, sample chamber, beam stop, and support structure were built

and a new detection system installed. The new beam tube is made of

two cylindrical aluminum sections lined with a lithiated polymer. The

sample chamber was also fabricated from aluminum and lined with

lithiated polymer, and may be evacuated to minimize the number of

neutrons scattered and absorbed by air. The beam tube and sample

chamber assembly is suspended from the aluminum support structure.

The detection system consists of a 40 % efficient germanium detector 

(resolution 2.0 keV at 1332.5 keV) and bismuth germanate Compton suppressor. The detection 

system shield consists of lead surrounded by borated and lithiated polyethylene, placed on a

table attached to the support structure. The new, more 

compact beam stop is welded to the support structure.

Captured gamma-ray photopeaks from H, B, C, N, Na, Al,

Fe, Ge, I, and Pb in the background spectrum were either of 

lower intensity or eliminated with the new PGAA instrument. 

The improved detection system, positioned closer to the 

ample, increased element sensitivity by 5 % to

CSTL researchers have a new 

thermal neutron prompt γ-ray 

activation instrument for use 

that was designed to provide 

greater sensitivities, better 

detection limits, and much 

lower background radiation. 

PGAA limits of detection have

been greatly reduced compared 

with those of the original

instrument due to reduced γ-ray

backgrounds and increased 

element sensitivities. 

s 50 %. 

Outreach Activities in Thermometry 

D.C. Ripple, K.M. Garrity, C.W. Meyer, G.F. Strouse, W.L. Tew, and C.D. Vaughn (836) 

The CSTL Thermometry Group strives to maintain world leadership in

thermometry, and to provide our users with all of the tools to attain

traceability to NIST standards. Research activities alone cannot achieve 

this goal. Outreach activities by the Thermometry Group provide visibility of 

our work, a mechanism for training and education of users, and a forum for 

cooperation and exchange of ideas with other scientists. By sponsoring 

conferences and presenting workshops, the CSTL Thermometry Group

educates industrial users and fosters communication within the field of

thermometry. In FY2003, the Thermometry Group sponsored the premier 

conference on thermometry, the 8th International Temperature 

Symposium. The proceedings of these Symposia are valued

for their documentation of the state of the art in thermometry. 

We also provided training to industry, through a workshop for

the ASTM committee on Petroleum Products and through 

workshops at the NIST campus in Gaithersburg. Because of 

In October 2002, CSTL 

scientists sponsored the 

International Temperature 

Symposium, which is held 

once a decade. 

126
our outreach activities, the Thermometry Group is world-renowned as a source of thermometry 

expertise. Numerous non-NIST laboratories have attained a level of competence in thermometry 

that is commensurate with many foreign National Metrology Institutes, partly because of ready 

ccessibility to methods established at NIST. 

ng and Measurement Assurance for the International Temperature Scale 

.F. Strouse and D.C. Ripple (836) 

a s

mprove the international acceptance 

inty claims by industry. 

g

e proficiency test and measurement assurance demands of the U.S. temperature community.

a

Proficiency Testi

of 1990 (ITS-90)

G

An increasing number of companies are seeking 

accreditation through NIST’s National Voluntary

Laboratory Accreditation Program (NVLAP) for ITS-90

Standard Platinum Resistance Thermometer (SPRT)

calibration services with uncertainty claims that are 

smaller than those of many National Metrology 

Institutes (NMIs). In order for NVLAP to grant 

and for those companies to gain acceptance in the global

marketplace, validation of the submitted uncertainty claims is

critical. For that purpose, the CSTL Thermometry Group

developed a tiered proficiency-testing program and service

that is designed with several levels of proficiency testing that

are commensurate with the submitted uncertainty claims. At

the lowest uncertainties, the level of scrutiny is comparable 

to that which an NMI undergoes for international acceptance.

The newly developed symbiotic relationship between the

CSTL Thermometry Group and NVLAP in the arena of

proficiency testing for ITS-90 SPRT calibration services will i

ccreditation to those companie

of NVLAP accreditations and uncerta

A selection matrix, with specific cutoff values 

based on an analysis of the uncertainties of 

numerous NMIs, is used in conjunction with the 

submitted uncertainty claims to determine the

appropriate proficiency test. The proficiency 

tests range from calibration of a single SPRT 

(large uncertainty claims) to a combination of a 

measurement assurance program (three SPRTs) 

and the direct comparison of fixed-point cells 

(NMI level uncertainty claims). A prototype 

NVLAP proficiency test was performed with a

U.S. company to test the new service, with

results shown. The new program, created and 

implemented within the CSTL Thermometry

Group includes protocols and report forms designed to maximize confidence in the results and 

transparency of the interpretation. CSTL continues to be the only laboratory capable of meetin

CSTL scientists have developed 

proficiency testing that provides 

the concrete evidence necess

for objective evaluation of

ary

a

laboratory’s capabilities. 

The design and implementation

of the NIST proficiency te

program in thermometry

assists industry in achieving 

accreditation and internati

acceptance, and furthers

confidence in the quality o

sting

onal

f

industrial measurements. 

-4

-2

0

2

4

0 200 400 600 800 1000

∆T, mK

Temperature, K

SPRT 1021

SPRT 1022

Sample results of a proficiency test. ∆T is 

equal to the participant calibration results 

minus the NIST results; error bars indicate

the combined expanded uncertainty (k=2) 

of the comparison.

th

127
A New Noninvasive Approach to the Measurement of Phase Equilibrium 

.J. Bruno (838) and W.C. Andersen (FDA) 

ontaining

mixtures, important industrially yet challenging b termolecular association. 

a.

nts were unsuccessful.

he new approach made the measurement possible.

dards for Thermal Analysis

.G. Archer (838)

l

rials for

alibration of TA instruments and for validation of standard test methods. 

T

Accurately describing the phase behavior of

mixtures, including those that are reactive, corrosive

or toxic, is crucial for optimizing large and smallscale chemical separations in the chemical and 

petrochemical industries. CSTL scientists have 

developed a new noninvasive approach

incorporating infrared spectroscopy for composition 

determination and a vapor entraining magnetic rotor that eliminates the need for recirculation 

pumps. The new apparatus measures phase equilibrium without disturbing the mixture being

measured. The new apparatus also permits observation and quantitation of any intermolecular 

complexation, and the novel metrology eliminates the need to withdraw any mixture samples from 

the measurement vessel. The instrument has been used for the phase equilibrium measurement 

of two systems: carbon dioxide + butane (to assess the capabilities of the instrument) and R-134a

+ polyolester lubricant (required for process optimization in the refrigeration/air conditioning

industry). Currently, the apparatus is being used for the measurement of amine c

CSTL scientists developed a 

noninvasive method to measure the

phase behavior of mixtures crucial 

for optimizing large and

ecause of in

Two unique and novel features provide the 

noninvasive approach. First, the composition 

measurement is based upon mid-range infrared 

spectroscopy (FTIR), with probes coupled to an

equilibrium vessel with fiber optic cables. The

compositions of the vapor and liquid phases of a 

mixture at equilibrium are determined from the 

spectra, independently and nearly simultaneously, at a particular temperature and pressure.

Moreover, any intermolecular complexation can be observed and measure

Second, the approach to equilibrium is driven by a new vapor entraining

magnetic rotor that eliminates the need for recirculation pumps. The novel

metrology eliminates the need to withdraw mixture samples from the

measurement vessel for any reason. Measurements on the refrigerant + 

lubricant mixture were needed by the U.S. Department of Energy, and

previous measurements using conventional instrume

small-scale

chemical separations. 

The new apparatus, designed and 

constructed by CSTL scientists, measures

phase equilibrium without disturbing the 

mixture being measured and has been used

for the following systems: CO Rricant; and amine 

2 + butane;

134a + polyolester lub

containing mixtures. 

d by the spectr

T

Measurement Stan

D

Thermal Analysis (TA) is a family of measurement

methods that follow a characteristic property (heat, mass, 

permittivity (dielectric), modulus, or others) of a material as 

temperature is varied. Thermal analysis methods are

widely used in research and in production analysis in

industries including pharmaceutical, polymer, chemical, aerospace, semiconductor and safety 

engineering. The widespread use of TA methods requires a measurement infrastructure that

ibration and standard test methods. This infrastructure requires at least two 

components: consensus documentary standards and reference mate

CSTL scientists provide 

leadership in the dev

of the measurement 

elopment

includes ca

c

This year we created and managed an Interlaboratory Test Study to determine the 

precision of this test method and to explore some of the confounding factors that 

infrastructure for TA methods.

128
affect interlaboratory precision. Volunteer laboratories 

were enlisted; materials were selected and tested at 

NIST and then distributed, with directions, to the

volunteer laboratories. The study was conducted in 

two parts. One group was given a sample of 1-phenyl1H-tetrazole-5-thiol, a thermally unstable mercaptan. 

The second group was given portions of a commercial 

sample of 2-butanone peroxide, a thermally unstable free-radical initiator used in the polymer 

industry. The results from 15 volunteer laboratories have been collected. These results will be

analyzed statistically and the appropriate research report will be completed. Also, the 

temperature and enthalpy of fusion of a sample of bismuth have been completed with hightemperature adiabatic calorimetry. These measurements will be the basis of the certification of 

RM 2235, bismuth for calibration of differential scanning calorimetry. 

Ionization Gauges

.J. Abbott (836); P. Mohan (NPL India)

P s directly to

th ar

rer’s lit

dependence of the gauge 

S

Improved Vacuum Transfer Standards –

P

CSTL’s goal is to disseminate NIST’s realization of the

our customers and into readily available commercial

gauges. However, commercial vacuum transfer

gauges are unstable and hamper efforts to effectively 

disseminate NIST’s uncertainties to our customers. In

recent years, a new and improved hot cathode 

ionization gauge has become commercially available. 

The sole manufacturer claims improved stability over

d-Alpert (BA) ionization

gauge by up to a factor of 10, thereby making it a superior

candidate as a high-vacuum transfer standard below 10

ascal at high vacuum level

at of a conventional Bay

-4 Pa. 

However, in the course of two recent rounds of calibrations at

NIST, several of these gauges have shown far worse stability

(calibration shifts as large as 10 % at the lowest measurable

pressures) under certain operating conditions than is reasonable to

erature. Furthermore, the 

response of the gauge

seems to depend strongly 

on its history of gas 

exposure. Our work 

confirms the history

expect from the manufactu

ASTM International Standard E967 

Standard Practice for Temperature 

Calibration of Differential Scanning 

Calorimetry and Differential Thermal 

Analyzers was revised and approved 

by consensus. 

CSTL investigators have 

documented the effect that ionic

pumping has on the stability of 

Bayard-Alpert (BA) gauges which 

will lead to reduced uncertainty in

high vacuum metrology. 

The gauge response p

to exposure and after 

exposure differed by

to 10% at 10

rior 

 up 

ufacturer 

expectations. 

-6 Pa of 

nitrogen, over three times 

higher than man

The change in gauge response

as a result of ionic pumping. The 

gauge was conditioned at 10-2 Pa

for 3 h on August 14, 2003. The

before and after conditioning 

data differ by as much as 10 % at 

the lowest calibration pressures. 

Nitrogen Calibration Pressure 

40

41.0

41.5

42.0

42.5

43.0

43.5

44.0

44.5

45.0

Gauge Sensiti

vity (Pa-1)

1.0E-06 1.0E-05 1.0E-04 1.0E-03 1.0E-02 1.0E-01

.5

October 3 Routine Calibration

August 13 Before Conditioning

n

August 15 After Conditioning 

August 18 Routine Calibration 

September 9 Routine Calibratio

129
response and identifies ionic pumping of gas into the gauge walls as the source of the instability. 

It also suggests a simple method of stabilizing the gauge’s response. We have established that

ionic pumping of gas by components internal to the gauges is responsible for the shifts in

calibration that have been observed. The pumping speed of the gauge was found to be greatest

just after a high temperature bake, a common procedure used to achieve high vacuum. It was 

also found that the gauge could not pump gas indefinitely, but rather became saturated after

exposure to a pressure of 10-2 Pa for three hours. 

Understanding the effect of ionic pumping on the stability of these gauges will lead to reduced 

uncertainty in high vacuum metrology, important to industrial process control, secondary 

laboratory calibrations, and international comparisons of high vacuum standards. Additionally,

these results provide important information to the manufacturer for mechanical re-design and/or

optimizing operating voltages of the gauge to minimize ionic pumping. 

Materials Evaluation and Database Tabulation for Fundamental Parameters and 

Algorithms Used For Quantitative Electron Probe Microanalysis 

J.T. Armstrong (837) 

In recent years, much work has been done to better understand the physical processes 

underlying electron probe microanalysis (EPMA) and improve the correction procedures

employed for quantitative analysis. Today there are literally dozens of different combinations of 

physical parameters and correction schemes in use by the active laboratories involved in

quantitative EPMA, and contained in the software packages of the various commercial 

manufacturers of x-ray analysis instrumentation. 

Carelessness has to an extent settled into the EPMA 

community, in that exactly which parameters and

algorithms have been employed to calculate elemental

concentrations is seldom reported (nor is the raw data

typically published). Different implementations of 

reputedly the same correction procedure can yield

significantly different results – either because of errors in the programming or undocumented 

changes/simplifications in equations and/or parameters. In some commercial systems, the 

corrections employed in the software are proprietary information and unknown to the user. As a

result it has become impossible to directly compare analytical results from different laboratories 

without adding a significant uncertainty due to the lack of

knowledge of how the data has been manipulated. Few

laboratories and no current commercial systems make any 

attempt to quantify the magnitude of this uncertainty in their 

error budget, although there is abundant evidence that it is 

often considerably larger than the reported uncertainties in

the measurements themselves. This work is part of an

ongoing effort to attempt to regularize, standardize and

improve the accuracy of EPMA data reduction to be 

comparable to the level of measurement precision. 

CSTL scientists have regularized, 

standardized, and improved the 

accuracy of EPMA data reduction 

to be comparable to the level of 

measurement precision. 

The parameters tabulated and 

evaluated were incorporated into 

the NISTZAF and TRYZAF 

correction programs and are 

available in text and spreadsheet 

files and have evaluative

comparisons with previously

used data. 

To that end, we have tabulated new sets of the fundamental parameters of use in quantitative

EPMA, based on the most recent experimental measurements and theoretical developments,

including data extracted from and traceable to the NIST Physics Laboratory Physical Reference

Database. In many cases, these values are significantly different from values currently in

common use by the EPMA community. The parameters tabulated and evaluated during the past

year were incorporated into the NISTZAF* and TRYZAF* correction programs. They include

ICRU tabulated mean ionization potentials, the mass absorption coefficients for K-alpha, L-alpha,

L-beta, and M-alpha lines between 100 eV and 50 keV derived from the NIST XCOM and NIST 

130
FFAST databases, the fluorescence yields compiled most recently by Hubbell, and the new x-ray 

line and edge energies of Deslattes. These tables are available in text and spreadsheet files and

have evaluative comparisons with previously used data. We have demonstrated that accurate

correction algorithms exist to predict the variation of x-ray intensity as a function of beam energy.

These methods can be used to accurately determine particle and layered specimen composition

(and thickness) without the need to independently standardize at each beam energy. They can

also assist in improving the accuracy of commercially popular “standardless analysis” procedures. 

*NISTZAF and TRYZAF allow the analyst to calculate concentrations and factors (atomic number

(Z), absorption (A), and fluorescence (F)) based on the k ratios and instrumental parameters. 

Development of a New Database for the Simulation of Electron Spectra for Surface 

Analysis (SESSA) 

C.J. Powell; W.S.M. Werner and W. Smekal (Contractors) 

A new NIST database has been 

developed for applications in Augerelectron spectroscopy (AES) and X-ray 

photoelectron spectroscopy (XPS), the 

two techniques most commonly used for 

surface analysis. The new database 

provides data for many parameters

needed for the quantitative interpretation 

of AES and XPS spectra. The parameters 

include: differential inverse inelastic mean

free paths, total inelastic mean free paths,

differential elastic-scattering cross 

sections, total elastic-scattering cross 

sections, and transport cross sections for 

both AES and XPS; Auger-electron

lineshapes, Auger-electron backscattering

factors, electron-impact ionization cross 

sections, and fluorescence yields for AES; 

and photoionization cross sections,

photoionization asymmetry parameters,

and photoelectron lineshapes for XPS. It 

also simulates spectra for multi-layered thin-film samples (with layer compositions and

thicknesses specified by the user) and measurement conditions specified by the user. The

simulated spectra can then be compared with experimental measurements, and the film

thicknesses and compositions adjusted to find maximum consistency between the simulated and

measured spectra. Most quantitative analyses by AES and XPS have been previously based on

the implicit assumption that the specimen is homogeneous 

over the sampling depth for the measurements or that the 

sample morphology is limited to one of several relatively 

simple morphology classes (and for which the effects of

elastic scattering were neglected).

Plots of a function of Si 2p photoelectron intensities (I)

for a 4 nm SiO2 film on a Si substrate as a function of

1/cosθ, where θ is the electron emission angle with

respect to the surface normal. The open circles show

experimental measurements, the solid line shows 

results from SESSA, and the dashed line shows the 

result obtained if elastic scattering is neglected. 

SESSA will enable improvements 

in the accuracy of quantitative 

AES and XPS analyses of multilayered structures. 

131
Evaluation of Electron Elastic-Scattering Cross Sections 

C.J. Powell; A. Jablonski (Polish Institute of Physical Chemistry); F. Salvat (Univ. of

Barcelona)

Differential cross sections for the elastic scattering of electrons by atoms are needed for modeling

the transport of electrons in materials by Monte Carlo 

simulations and other means. Such transport 

calculations are needed to improve the accuracy of 

surface analyses, bulk analyses, and thin-film 

analyses of inhomogeneous samples by the 

techniques of Auger-electron spectroscopy (AES), Xray photoelectron spectroscopy (XPS), electronprobe microanalysis (EPMA), and analytical electron

microscopy (AEM). The cross sections are also needed for similar transport calculations in other 

applications including radiation dosimetry, radiation therapy, radiation processing, radiation

protection, and electron-beam lithography. Since cross sections have been measured only for a

limited number of atoms and electron energies, it is necessary to make use of calculated cross

sections in the transport calculations. 

CSTL scientists examined the 

extent to which three quantities 

derived from Differential Cross 

Sections (DCS) varied depending 

on whether they were obtained 

from the TFD or DHF potential.

An extensive analysis was made of differential cross sections (DCSs) for the elastic scattering of

electrons by neutral atoms that were derived from two commonly used atomic potentials: the

Thomas-Fermi-Dirac (TFD) potential and the Dirac-Hartree-Fock (DHF) potential. DCSs from the

latter potential are believed to be more accurate. We 

compared DCSs for six atoms (H, Al, Ni, Ag, Au, and Cm) at

four energies (100 eV, 500 eV, 1000 eV, and 10 000 eV) 

from two NIST databases in which DCSs had been obtained 

from the TFD and DHF potentials. While the DCSs from the 

two potentials had similar shapes and magnitudes, there 

could be pronounced deviations (up to 70 %) for small 

scattering angles for Al, Ag, Au, and Cm. In addition, there were differences of up to 400% at 

scattering angles for which there were deep minima in the DCSs; at other angles, the differences 

were typically less than 20 %. The DCS differences decreased with increasing electron energy.

DCSs calculated from the two potentials were compared with measured DCSs for six atoms (He,

Ne, Ar, Kr, Xe, and Hg) at energies between 50 eV and 3 keV. For Ar, the atom for which

measured DCSs are available over the largest energy range, there was good agreement between 

the measured DCSs and those calculated from the TFD and DHF potentials at 2 keV and 3 keV,

but the experimental DCSs agreed better with the DCSs from the DHF potential at lower 

energies. A similar trend was found for the other atoms. At energies less than about 1 keV, there 

were increasing differences between the measured DCSs and the DCSs from the DHF potential. 

These differences were attributed to the neglect of absorption and polarizability effects in the 

calculations. 

The atomic differential cross 

sections were empirically useful 

for simulations of electron

transport in solids for electron 

energies above about 300 eV. 

We also examined the extent to which three quantities derived from DCSs varied depending on 

whether the DCSs were obtained from the TFD or DHF potential. First, we compared calculated

and measured elastic-backscattered intensities for thin films of Au on a Ni substrate with different 

measurement conditions, but it was not clear whether DCSs from the TFD or DHF potential 

should be preferred. Second, we compared electron inelastic mean free paths (IMFPs) derived

from relative and absolute measurements by elastic-peak electron spectroscopy and from

analyses with DCSs obtained from the TFD and DHF potentials. In four examples, for a variety of 

materials and measurement conditions, we found differences between the IMFPs from the TFD 

and DHF potentials ranging from 1.3 % to 17.1 %. Third, we compared mean escape depths for 

two photoelectron lines and two Auger-electron lines in solid Au obtained using DCSs from the

TFD and DHF potentials. These mean escape depths varied from 4.3 % at 70 eV to 0.5 % at

2016 eV at normal electron emission, and became smaller with increasing emission angle.

132
Although measured DCSs for atoms could differ from DCSs calculated from the DHF potential by

up to a factor of two for electron energies less than 1 keV, we found that the atomic DCSs were

empirically useful for simulations of electron transport in solids for electron energies above about

300 eV. This unexpected result appears to be due to the smaller absorption correction (i.e.,

smaller total inelastic scattering cross sections) in the calculated DCSs for solids than for the

corresponding atoms. The atomic DCSs could also be useful for energies down to at least 200 eV

if relative measurements were made. 

Development of Critical Benchmark Standards and Methods for the Metals Industry 

J.R. Sieber, A.F. Marlow, M.R. Winchester, L.L. Yu, T.A. Butler (839); R.M. Lindstrom (837); 

M.P. Cronise, and C.N. Fales (TS) 

Metals Industry laboratories need assistance with

certified reference materials, improved analytical 

methods and uncertainty estimates. NIST and 

ASTM International have a long-standing 

partnership to develop SRMs and

analytical methods. Collaboration is 

even more important now that NIST is implementing a quality system in compliance

with ISO/IEC 17025, General requirements for the competence of testing and

calibration laboratories. To meet the challenges of industry-wide compliance with ISO/IEC 

17025, it is crucial to have effective industry standard test methods that make efficient use of

certified reference materials. This fiscal year, the effort on behalf of the metals industry was

diversified to include six SRMs for metals and alloys, including: SRM 2426, Galvalume®, Al-Zn 

Alloy; SRM 2696, Silica Fume; SRM 2453, H in Titanium; two SRMs for related materials, and 

participation in the development of a new ASTM International Standard Method of Test for Nibase alloys. The new test method for Ni-Co alloys is of critical importance for alloy production

control and product quality. With the NIST input, the new method provides options for calibration

of X-ray fluorescence (XRF) with fewer reference materials. All of these accomplishments benefit 

the titanium, steel and aluminium segments of the metals industry, the petroleum industry, the

mining industry and their customers in aerospace, automotive, semiconductor, and construction 

industries. None of this could be accomplished without the expertise and close cooperation of 

leading metals producers. 

CSTL researchers in collaboration 

with industry have produces several 

new SRMs that will benefit the 

customers in aerospace, automotive, 

semiconductor, and construction 

industries. 

The NIST contribution to a new ASTM XRF method for Ni-base alloys gives industry labs the 

option of using a full fundamental parameters (FP) calibration. FP methods require fewer 

calibration standards than conventional XRF methods for calibration of matrix corrections. This is 

one way to reduce demand for reference materials while concurrently broadening the scope of

the method. The wide range of reference materials 

developed this year, demonstrates the breadth of the

US metals industry, its suppliers, and its customers. It 

also demonstrates the need to renew existing SRMs 

that have gone out of stock. Feedback from ASTM 

International makes it clear that the industry requires a 

steady supply of both new and existing SRMs to 

maintain analytical methods that satisfy its own and its 

customers’ high expectations for product quality. 

These accomplishments benefit the

titanium, steel and aluminium 

segments of the metals industry, the 

petroleum industry, the mining 

industry and their customers in 

aerospace, automotive, 

semiconductor, and construction 

industries. 

133
SpectroML—An Extensible Markup Language for UV-Visible Spectroscopy Data 

A.D. Nguyen, A. Arslan, M.V. Smith, J.C.Travis, and G.W. Kramer (839)

The interchange and storage of analytical chemistry data has long been hampered by multiple,

incompatible data formats. Over the past 15 years, several different interchange formats have 

been developed that allow data generated by different vendor systems of a given technique to be

exchanged. For example, using JCAMP-DX, users of

most commercial FT-infrared and NMR systems can 

interchange their respective data. However, in the last

few years, the emergence of platform and application

independence through the Internet has advanced the 

possibilities for data interchange beyond the capabilities 

of these protocols. 

CSTL scientists developed an 

XML-based mechanism for 

instrument-to-instrument, 

instrument-to-application, and 

application-to-application data 

interchange called SpectroML. 

Today’s analytical chemists need not only to interchange data generated within a specific 

technique, they need to interchange, import, export, store,

and combine all their data from multiple sources at multiple

sites. Users are now no longer content with just having 

“pictures” of their data (i.e., data plots). They need real

access to their data from within a report itself—they want to

be able to expand plots, re-integrate peaks, overlay spectra, 

etc. without having to revert to the instrument or computer 

system that generated the original data. And, they don’t want 

to have to purchase, learn, or even load proprietary programs to do this. In industrial settings,

experimental data belong to and must be accessible to the entire organization, not just the 

laboratory personnel that generate it. Proprietary data formats and existing interchange

programs simply cannot fill such corporate needs for data accessibility. 

AnIML is based on a 

hierarchical model that calls 

for the initial development of a 

core markup language 

containing the elements 

common to all applicable 

analytical techniques. 

We have created an extensible markup language (XML) for UV/visible data called SpectroML and

are using it to interchange data between our spectrometers and our optical filters database.

SpectroML is now an integral component in certification and recertification process for optical 

filter SRMs. We are working with the ASTM Subcommittee E13.15 on Analytical Data

Management, representatives from instrument companies, and the IUPAC committee responsible

for JCAMP-DX to create a unified XML-based approach for interchanging molecular spectrometry 

and chromatography. Based in part on SpectroML, this effort is being called the Analytical

Information Markup Language (AnIML). The development of AnIML as a universal molecular 

spectrometry and chromatography mark up language will revolutionize data interchange, 

eliminate long standing barriers to data exchange, enable new data manipulation software, 

facilitate new web-based notions for data usage, and permit the development of realistic schemes 

for long-term data archival. When coupled with parallel efforts in developing XML approaches to 

the manipulation of other chemical data, it will pave the way for the fully electronic chemistry data 

archive, where any known chemical fact or information is but a simple query away on your

wireless Personal Digital Assistant from anywhere in the world. 

134
Molecular Simulation of Alkylsilane Stationary Phases in Liquid Chromatography 

K.A. Lippa and L.C. Sander (839); R.D. Mountain (838) 

Shape-selective polymeric alkylsilane

stationary phases (typically C18 chains) are 

routinely employed over the more common

monomeric alkylsilane stationary phases in 

reversed-phase liquid chromatography (RPLC) to improve the separation of geometric isomers of

constrained solutes, such as polycyclic aromatic hydrocarbons, carotenoids, steroids and

polychlorinated biphenyls. Critical to describing such shape-selectivity processes in RPLC is the 

elucidation of the molecular-level structural features of the alkylsilane stationary phase that

promote such separations. Previous molecular dynamic simulation models that illustrate the

structure and dynamics of alkyl

chromatographic stationary phases have been 

focused on only monomeric-type materials.

We have investigated the molecular dynamics 

of chromatographic models that represent both

monomeric and polymeric stationary phases 

with alkylsilane lengths, surface coverages, 

bonding chemistries and temperature

conditions that are typical of actual materials 

prepared in the laboratory. 

CSTL scientists found that alkylsilane 

chain structural analyses are consistent 

with previous experimental observations.

The resultant structural features of the 

alkylsilane chains (i.e., length, conformational 

order, gauche dihedral angle defects) of our 

simulation models were related to the 

observed shape-selectivity properties of

corresponding LC stationary phases. The 

structural characterization of these

computational models are consistent with

previous experimental observations: 1) alkyl 

chain order increases with increased surface 

coverage; 2) monomeric and polymeric phases

with similar surface coverages give rise to

similar degrees of alkyl chain order; 3) longer 

chain length phases exhibit significantly more

order than C18 phases; and 4) alkyl chain

ordering is significantly enhanced at lower 

temperature. In addition, a significant portion 

of the alkyl chain closest to the silica substrate

surface is disordered and the opposite end is 

most ordered. 

For simulation models that represent RPLC columns that

have been characterized as highly shape-selective (through 

use of the SRM 869a Column Selectivity Test Mixture), a

significant region of alkyl chain order with primarily trans

dihedral angle conformations is observed. This is consistent 

with the view that the alkyl chains comprising the polymeric

stationary phase contain a series of well-defined and rigid

slots in which shape-constrained solutes can penetrate and 

hence be selectively retained. The results of this recent 

simulation work have promoted the synthesis of low- to moderate-surface coverage polymeric 

Top and side view snapshots of 

molecular dynamic simulated LC phases 

at experimentally relevant surface 

coverages (μmol/m2

). Alkylsilane chains 

are displayed in gray (C), blue (Si) and 

red (O). The silica surface is displayed in 

red (O) and yellow (Si). Hydrogen is not 

displayed for clarity.

The application of molecular 

dynamic simulation techniques 

for the design of future RPLC 

phases will enable the analyst to 

intelligently design surfacemodified materials that possess 

specific structural characteristics

that will result in a particular 

retention mechanism.

135
alkylsilane RPLC stationary phases that are anticipated to possess unique shape-selectivity 

behavior. 

Low Gas Flow Measurement Assurance Program 

J.D. Wright and G.L. Kline 

A Measurement Assurance Program (MAP) 

ended in September 2003 that compared the gas 

flow standards of Department of Defense (DoD) 

laboratories to each other and to the national gas 

flow standards at NIST. Two sets of four laminar 

flowmeters (Molblocs manufactured by DH 

Instruments) were used as the transfer standards

(TS) during the MAP. The 8 transfer standards

were calibrated between 0.04 and 30 L/min on

more than 10 occasions at NIST, over the 2-

year period of the MAP, and were calibrated by 

the participating labs as well. The participating

labs were the Air Force Primary Standards 

Laboratory, the Army Primary Standards 

Laboratory, the Marine Corps Logistics 

Laboratory, and DH Instruments in Phoenix,

Arizona. 

CSTL scientists compared the DoD 

and DHI labs with each other and to 

the NIST standards with the purpose 

of evaluating the Molbloc laminar 

flowmeters for calibration stability, 

gas specie effects, and suitability as 

working or reference standards to 

calibrate other flowmeters. 

The uncertainty of mass flow measurements

made with the transfer standard (including 2-

year reproducibility) were 0.13% to 0.23% on 

flow range of these data. Differences between 

individual labs and NIST were as large as 3%,

but in general, values of 0.6% or less were 

observed. The flow differences at one of the 

tested flows (1 L/min) are graphically 

presented to the left. In this plot, made with

data collected independently from two

flowmeters at the same flow, separation

between labs (A, B, C, etc.) along the

diagonal represents systematic 

differences. The NIST reference value

at (1,1) has error bars proportional to

the 2-year reproducibility of the transfer

standard. The transfer standard 

reproducibility in each lab is shown with 

error bars as well. The differences

between labs were generally within the uncertainty expectations (0.16% to 0.55%), but in several

cases, changes in the participants’ facilities or procedures were recommended that would 

improve their calibration results. The DoD participants used piston provers, and significant

differences in data scatter were discovered depending on the design of their provers.

A Youden plot for the 1 L/min 

comparison data, all labs. 

The transfer 

standard 

with its 

laptop data 

acquisition 

computer. 

The comparison showed where uncertainty improvements could be made, demonstrated flow 

measurement proficiency, and established flow traceability from the U. S. national standards to

the participants. This improves the accuracy of flow measurements made by end users and 

136
assures that the participating labs’ 

uncertainty specifications are met

under all the actual, dynamic 

conditions of measurement. 

Our DoD sponsors will make the transfer standard 

available for other laboratories to use in their efforts 

to maintain proficiency and traceability to NIST. 

137
138
139

10. Microelectronics 

CSTL supports the microelectronics industry by providing the

necessary physical standards for process control of parameters such

as temperature and humidity. CSTL also provides reference materials 

to facilitate quality control in manufacturing, as well as providing 

reliable kinetic and thermophysical property data to facilitate process 

modeling, to meet critical industry needs. CSTL research remains on 

the cutting edge in order to provide dramatically improved measurement tools to keep advanced 

U.S. microelectronics manufacturing globally competitive. 

Development of Phosphorus Ion Implant in Silicon Depth Profile Standard 

D. Simons (837), R. Paul (839), W. Guthrie and J. Lu (898)

This project provided the U.S. semiconductor industry with a means to measure the implanted

dose of phosphorus in silicon with low uncertainty. The shrinking size of microelectronic devices 

based on silicon technology has lead to narrower tolerance requirements for implanted doses of 

electrically active dopant species, and more attention toward confirmation of electrical models of

device performance. Both of these developments require accurate determination of dose values

by secondary ion mass spectrometry (SIMS), the analytical method of choice for dopant

measurements. Previously NIST had developed and

issued Standard Reference Materials for calibration 

of SIMS measurements of implanted doses of boron 

(SRM 2137) and arsenic (SRM 2134) in silicon. The 

specific need for a certified reference material for 

phosphorus, the third major dopant in silicon, was 

demonstrated by an interlaboratory round-robin exercise that showed nearly a factor of two

disparity in measured dose values among participating laboratories when they used their own inhouse phosphorus reference materials. That need has now been satisfied through the availability 

to the U.S. semiconductor industry of SRM 2133 – Phosphorus Implant in Silicon Depth Profile 

Standard, issued in April, 2003. 

The phosphorus dose in SRM 2133 was 

certified by radiochemical neutron 

activation analysis with an expanded 

relative uncertainty of 1.7 %. 

All known sources of uncertainty were considered, including a small spatial heterogeneity among

samples. SRM 2133 should meet the needs of the U.S. semiconductor industry for many years 

based on the projections of the latest edition of the International Technology Roadmap for 

Semiconductors. 

Depth (μm)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

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m3

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SRM 2133 

100 keV 

P→Si 
Proximity Effects of Lightpipe Thermometers in Rapid Thermal Processing Tools 

K. G. Kreider and W. A. Kimes (836); D. P. DeWitt, B. K. Tsai (PL) 

Lightpipe radiation thermometers (LPRTs) are used as

temperature-monitoring sensors in rapid thermal

processing (RTP) tools for semiconductor fabrication.

These LPRTs can yield accurate temperature

measurements when they are calibrated in situ or

calibrated ex situ and used with a suitable model to

correct for surface emissivity and chamber irradiation 

effects. Wafer temperature measurements are 

frequently performed in a highly reflecting chamber to

obtain a near-unity effective emissivity of the wafer. However, the sapphire lightpipe tip has a low

reflectivity (high absorptivity) that enhances radiation heat transfer from the target region. This

low reflectivity causes a depression in

the wafer temperature, degrading the 

thermal uniformity necessary for

fabrication of high-quality 

semiconductor devices. This project

was initiated to quantify the effect of 

lightpipe proximity to the wafer 

temperature measurement and to 

provide a model for use with 

commercial tools. 

Rapid thermal processing (RTP) 

is used for thermal oxidation and 

dopant annealing of silicon 

wafers. Accurate temperature 

measurements from commonly 

used lightpipe radiation 

thermometers (LPRTs) are 

critical to this process. 

140

To minimize disturbances in the 

reflectivity of the RTP chamber, small, 2

mm diameter, sapphire lightpipes are 

often the temperature sensor of choice.

Our experiments were performed to 

measure and model the effect of LPRT

proximity on the wafer temperature in

the NIST RTP test bed. We measured 

the spectral radiance temperature with 

the center lightpipe and compared

these measurements with the three

LPRTs at the mid-radius of the wafer and the thin-film thermocouple (TFTC) junctions of a NIST

calibration wafer. Depressions in the wafer temperature up to 25 °C with the lightpipe at 2 mm 

spacing were observed. A finite-element radiation model of the wafer-chamber-lightpipe was

developed to predict the temperature 

depression as a function of the lightpipe 

proximity distance and the chamber-wafer 

separation. The experimental results 

were compared with those from a model 

that accounts for lightpipe geometry and 

radiative properties, wafer emissivity and 

chamber cold plate reflectivity.

-25

-20

-15

-10

-5

0

600 700 800 900

Mid-radius Radiance Temperature / °C

Temperature Depression / °C

6mm

4mm

3mm

Temperature depression caused by an LPRT at the 

indicated distances from a wafer. Symbols are 

measured values. The solid and dashed lines 

represent values from the model for proximities of 6 

and 3 mm respectively. 

Technical results are reported in “Effects of

Lightpipe Proximity on Si Wafer Temperature in 

Rapid Thermal Processing Tools” AIP Conference 

Proceedings on Characterization and Metrology for 

ULSI Technology, 2003. 
Development of Plasma Process Monitoring and Diagnostic Techniques for the 

Semiconductor Industry 

M. Sobolewski and K. Steffens (836) 

Over a third of the steps in the fabrication of microelectronic 

devices use plasma deposition and etching approaches for surface

treatment and thin film deposition. Plasma models and diagnostics 

are now being implemented in process monitors and controllers to

improve processing flexibility and reliability. Plasma processing 

reactors have historically been designed and operated using 

empirical methods alone, but continued evolution of these tools 

requires a much greater 

reliance on process and reactor modeling. To obtain 

more reliable predictions of the spatial uniformity,

chemistry, and electrical properties of processing 

plasmas, further progress in model development and

validation is required. Also, to enable improvements in 

process control, a need exists to develop sensors that 

are compatible with the manufacturing environment. 

Model-based plasma 

process design and 

control is an important 

need identified in the

International 

Technology Roadmap

for Semiconductors

141

One major area of activity has been the development 

and testing of a model-based technique for in-situ, realtime monitoring of ion energy distributions and total ion

flux. The technique relies on radio frequency (rf) 

current and voltage measurements made outside of a 

plasma reactor, and thus it is well-suited for process 

monitoring applications in manufacturing where the use 

of invasive probes to measure ion energy or flux is not

practical. Recently, tests of the technique were

performed in argon and CF4 discharges. The tests 

included a complete sensitivity analysis which 

quantified the dependence of ion energies and other 

outputs on all required input parameters, allowing us to

rigorously determine the uncertainties in the outputs.

Also, recent experiments have demonstrated the 

application of the technique to real-time monitoring of 

process drift in an inductively coupled plasma 

reactor. The drift occurs due to the deposition of a 

conductive layer on the surface of the dielectric 

window adjacent to the inductive source. Changes in

the conductive layer produce large changes in ion

flux and ion energy which are accurately monitored in

real-time by the model-based technique. 

As model-based reactor design and process 

development become increasingly utilized, species 

density measurements and gas temperatures can

provide important input and validation for plasma

modelers. Spatial variations in plasma temperature

can cause spatial differences in gas density and 

reaction rates and can complicate the interpretation

of gas density measurements. The planar laserinduced fluorescence (PLIF) technique not only 

provides two-dimensional (2-D) density maps of Temperature maps of CF4 plasmas.

measured by

mass spectrometer

noninvasive

measurements

CF3

CF2

+

+

F+

CF+

0 20 40 60 80

ion energy (eV)

Ion energy distributions measured 

by the noninvasive, model-based 

technique (bottom) and by a mass 

spectrometer (top). 
important fluorocarbon radical species in dielectric etching plasmas, it also has been extended to

enable 2-D temperature mapping in fluorocarbon plasmas. Using PLIF of the CF radical,

temperature maps have been measured in CF4 plasmas as a function of pressure and power.

Temperature has been observed to vary spatially by up to 150 K in these plasmas. Temperature

increases with pressure and power and is lowest near the cooled electrode surfaces. In addition, 

the presence of a silicon wafer was found to increase the temperature throughout the plasma, 

especially when heat transfer between the wafer and the cooled electrode surface was poor. 

Thermophysical Properties of Gases used in Semiconductor Processing 

J. J. Hurly, K.A. Gillis, and M.R. Moldover (836) 

The semiconductor industry requires accurate process 

gas property information to develop and control

manufacturing processes such as gas control in 

chemical vapor deposition processes and the use of 

mass flow controllers. CSTL responds to these data 

needs by measuring and publishing the thermophysical 

properties of semiconductor process gases. These

measurements exploit novel, accurate, NIST-developed

acoustic techniques. The measured properties include 

the speed-of-sound, ideal-gas heat-capacity, density 

(equation of state), viscosity, and thermal conductivity. 

Representatives of the semiconductor industry have

identified the process gases, “surrogate” gases, and the

binary mixtures of process and carrier gases of highest

priority. They also established targets for the accuracy of the thermophysical property data that

are needed to model manufacturing processes and to control the process quality. Specific areas 

that will benefit from this work are chemical vapor deposition (CVD) processes and the calibration

of mass flow controllers (MFCs) using surrogate gases. As results are acquired, NIST 

disseminates them via the internet at the URL http://properties.nist.gov/semiprop. The database 

includes the heat capacity at constant pressure,

thermal conductivity, viscosity, and virial coefficients 

B(T) and C(T) that determine the pressure-densitytemperature relation. The diffusion coefficients for 

gaseous mixtures of process gases and carrier 

gases will also be included. 

142

This year a new viscosity measurement system for 

hazardous gases was completed and used to 

measure the viscosity of 9 gases. The acoustic 

viscometer also determines the speed of sound in

the test gas. We used this data to determine the 

ideal-gas heat-capacity to within 0.1 % and virial

coefficients that reflect each gas’s non-ideality. 

From the virial coefficients, we developed an

equation of state that predicts the gas’s densities to

within 0.1 %. 

Gases and Conditions for Viscosity Data. 

Gas Temperature

Range (K) 

Maximum 

Pressure, MPa 

He 298 3.3 

Ar 200 - 375 3.3 

N2 298 3.3 

C3H8 225 - 375 0.9 

SF6 298 1.8 

CF4 200 - 375 3.3 

C2F6 225 - 375 2.8 

N2O 225 - 375 3.4 

NF3 225 - 375 3.4 
Electron Probe Characterization of KNbO3 and NaNbO3 Single Crystals for use as 

Quantitative Microanalysis Reference Materials 

R. B. Marinenko (837), Z. Samardžija, S. Bernik, B. Malič, M. Čeh (Jožef Stefan” Institute, 

Jamova 39, SI-1000 Ljubljana, Slovenia)

Alkali niobates are being used as optoelectronics components due to their distinctive electrooptical, nonlinear optical properties. Many of the niobates are chemically complex and require

accurate chemical reference materials to aid in determining their “as processed” composition.

One area of specific interest is reference standards for electron probe microanalysis (EPMA) of

alkali niobates with the general formula K1-XNaXNbO3, a ferroelectric material with perovskite

structure. The ceramic material K0.5Na0.5NbO3 (KNN), a solid solution of the two alkali niobates,

is of major interest because of its high piezoelectric coefficients and moderate dielectric 

permitivity. Complicated fabrication can lead to undesirable multiphase ceramics; therefore, a 

careful characterization of the product is required on the micrometer scale to verify that the

desired composition has been produced. 

143

% Mass Fraction (1 σ uncertaity in parentheses) 

 KNbO3 NaNbO3 (0.30 % mf Ca) 

K Nb O (stoic.) Na Nb O (stoic) 

EPMA-WDS 21.56 (0.14) 51.54 (0.16) 26.90 14.08 (0.08) 56.53 

(0.20) 

29.09 

Nominal 21.72 51.61 26.67 14.03 56.69 29.28

KNN Anal. 9.97 (0.28) 19.98 (0.31) 60.31 

(0.41) 

9.74 (0.26) Form. K0.50Na0.49Nb1.00O3

Potassium and sodium niobate single crystals were evaluated with the electron probe 

microanalysis (EPMA) using wavelength dispersive spectrometers (WDS). Alkali metal migration

that usually occurs in materials exposed to the electron beam, was insignificant. The 

microheterogeneity determined from line traverses and random points was minimal with all 

analysis points being less than the expanded 3 σ uncertainty predicted from the x-ray counting 

statistics for each element. Quantitative analysis was also in agreement with the expected

stoichiometry of the crystals although a Ca impurity of 0.3 % mass fraction was found in the 

NaNbO3 specimen. 

The KNbO3 and NaNbO3 single crystals tested appear to be excellent reference materials for

characterizing KNN ceramics with EPMA on the micrometer scale. In addition, because no 

migration of the alkali elements were observed when these specimens were tested with a highenergy electron beam, these materials will make excellent K and Na reference standards for the

microanalysis of other alkali-containing materials.

Non-Contact Free Carrier Density Measurements for Compound Semiconductors 

J. E. Maslar and W. S. Hurst (836) 

Transport of free electrical carriers is central to the operation of all optoelectronic devices and

reliable measurement of the carrier properties is critical. Hall or capacitance-voltage 

measurements are traditionally used to obtain this information, but require electrical contact. This 

precludes the use of these techniques in situ during growth or processing and, typically, even on
actual device layers. Raman spectroscopy, as an optical 

technique that can be used for transport property

determination, does not suffer from these limitations. In

addition, it is non-destructive, spatially resolved, and can

be applied to a specific buried layer, which is sometimes

a problem for traditional electrical measurements. A number of issues are central to determining 

the accuracy and precision of this method, including the semiconductor material under 

investigation, the measurement system parameters, and the Raman spectral model used to fit the

measured spectra. NIST is systematically addressing such issues. The results of this 

investigation should facilitate the utilization of Raman

spectroscopy for spatially resolved, off-line

characterization as well as process monitoring and

control during film growth and subsequent patterning

processes. 

Accurate in situ methods are 

needed to quantitatively 

determine the electrical carriers 

in optoelectronic components 

144

Raman spectra of n-type doped GaSb, p-type GaSb,

n-type GaInAsSb, and n-type GaN were recorded to 

determine the carrier density. Modeling of the 

Raman spectra from the different materials also 

requires different spectral models. The spectral 

models for the Sb-based materials are based on a

zinc blend crystal structure and account for 

conduction band non-parabolicity, absorption of 

excitation radiation, and included multiple carrier 

types (due to multiple conduction band minima being 

occupied). The computer code for modeling the Sbbased materials has been written and all n-type Sbbased material spectra we have obtained have been

fit. These results have been compared to electrical 

measurements. From this comparison, it was 

determined that more sophisticated spectral models 

should be developed. The spectral models for the

N-based materials are based on a wurtzite crystal structure and do not need to account for 

absorption or multiple carrier types. Intensity (Arb. Units)

200 300 400 500 600 700

Raman Shift (cm-1)

 Measured Spectrum

 Fit Spectrum

L+

LLO

2O

The measured Raman spectrum for 

n-type GaSb. Fitting the data with a 

Raman spectral model results in a 

determination of carrier concentration

of about 6 x 1018 cm-3

Neutron Transmutation Doping of Compound Semiconductors 

R.M. Lindstrom (839) 

The addition of small amounts of selected elements to silicon 

and other materials controls the semiconducting nature of the 

material. Typically these elements are implanted at a high 

energy then annealed to more evenly distribute the dopant.

Doping of semiconductors can also be accomplished by using 

neutron capture, followed by beta decay, to transmute 

elements. New compounds are being explored, to exploit the 

experience gained in industry with silicon. 

The ability to dope GaN 

uniformly would be as 

useful for tuning the 

semiconductor properties 

of this promising material.

Transmutation of silicon by nuclear reactions has become well established for the production of

high-power rectifiers and other electronic devices. In this application, phosphorus is created by 

the capture of neutrons by 31Si to make 32Si, which decays to 32P. Because silicon is transparent 

to neutrons, the phosphorus dopant is created uniformly through a large Si crystal. 
The same process as used for elemental silicon is applicable to 1:1 compound semiconductors.

Recently, neutron transmutation doping (NTD) has been explored in two additional systems. In 

collaboration with North Texas State University and the NIST Center for Neutron Research, 

samples of Hg0.8Cd0.2Te films on Cd0.95Zn0.05Te substrates have been subjected to neutron

irradiation. The goal is to transmute both Hg to Au and Te to As. A first experiment showed that

transmutation is feasible. The results of this experiment were reported at the Narrow Bandgap 

Conference in 2003. To study the effects of irradiation quantitatively, additional samples have

been exposed to a pure thermal neutron flux over a range of fluences. 

Gallium nitride is of great current interest for high-temperature devices. The opportunity exists for 

uniform doping of this material, by transmuting both 69Ga and 71Ge to the corresponding Ge

isotopes. In collaboration with IEEL, a sample of bulk GaN was obtained from ATMI, Inc. and 

irradiated with 1.0 x 1018 neutrons/cm2

, for a calculated uniform doping of 2.5 x 1016 atoms Ge/g

Ga, or 1.3 x 1017 atoms Ge/cm3

. This material is has been returned to ATMI for characterization. 

High-Resolution Cavity Ring-Down Spectroscopy (CRDS) Measurements of Water Vapor 

J.T. Hodges and J.G. Cormier (836)

CRDS is being developed as a primary measurement standard enabling quantitative on-line

measurements of water vapor at trace concentrations 

≈1010 molecules cm-3 to 1013 molecules cm-3, a domain 

where gravimetric techniques are inapplicable. This 

research has applications in semiconductor

processing, as well as trace gas monitoring and 

analysis, and atmospheric research. It also supports a 

broad class of spectroscopic techniques that are

based upon the absorption of electromagnetic 

radiation by water vapor. 

145

In FY 2003, two sets of CRDS experiments covering

different spectral ranges were implemented. In the first system, a CW diode laser and a custom 

high-resolution CRDS apparatus were used to measure absolute line strengths, and line shapes 

for water vapor transitions in the near-IR. A long-term reproducibility of better than 0.3 % and

relative uncertainty less than 2 % in the 

determination of line strength was 

demonstrated. (See figure for a typical 

spectrum.) The effects of pressure

broadening and collisional narrowing of the

line shape were quantified and a spectral 

resolution on the order of 1 MHz was 

achieved, illustrating the high spectral 

resolution of the CRDS method. The second 

CRDS experiment considered the water

vapor absorption continuum in the mid-IR 

spectral range. The temperature

dependence of the continuum absorption 

coefficient was measured from –15 °C to 20

°C. This work exploited the high precision of

the mid-IR CRDS technique and access to 

NIST standards of humidity generation and 

measurement. A detection limit of 30 pmol 

mol-1 was demonstrated. Finally, to 

Trace amounts of water vapor 

occurring in bulk source gases or 

de-adsorbed from system surfaces 

can adversely effect important 

processes related to the growth 

and manufacturing of 

semiconductors, photonic devices 

and other micro- and nano-scale 

solid state systems. 

-2000 0 2000

20

25

30

35

40

45

50

-0.5

0.0

0.5

 data

 Galatry fit

 Voigt fit

106

*single pass loss 106*residual

Detuning (MHz)

Measured spectrum of H2O absorption

transition, CRDS measurements and best-fit 

theoretical line shapes (bottom), and fit 

residuals (top).
complement the mid-IR CRDS studies of the water vapor continuum, similar CRDS 

measurements of the water vapor continuum were also begun using the near-IR CRDS system. 

These studies suggest that near-IR CRDS measurements of the water vapor continuum may 

answer some important scientific questions regarding the role of water dimers in the radiation

absorption by atmospheric water vapor. 

Identification of Contaminants in Surface Films by Scanning Auger Microscopy

S.A. Wight (837)

146

he contaminate particles in the tinhe contamination films on the copper plate were identified as mixed hydrocarbon and oxide with

The surface specificity of Auger microscopy is ideally suited to identify contaminant films and 

particles on surfaces. In the electronics industry, as devices continue to shrink, scanning Auger 

microscopy is being more widely utilized to identify 

contaminates. NIST's analytical scanning Auger microscope 

(ASAM) has been applied to two specific measurement 

challenges. 1) It was used to identify the contaminant 

precipitates in tin-copper electrodeposited films on copper 

substrates. These films are an important step towards leadfree solders for the microelectronics industry. 2) The ASAM 

was used to identify and provide a maximum thickness for 

the contamination areas on a copper plate used for the

proposed new NIST capacitance standard

based on the charge of the electron. 

CSTL researchers use 

scanning Auger microscopy 

to identify contaminants on 

the surfaces of two 

technologically important 

materials for the 

semiconductor industry. 

20000

T

copper electrodeposited films were

identified as tin oxide precipitates by

scanning Auger microscopy. Prior to

analysis, the area of interest was carefully 

cleaned, by low energy ion etching, to

remove the ubiquitous organic

contamination that otherwise masks the 

composition of the particles. In the

cleaning process it was necessary to

strike a critical balance between removing

the oxygen and organic contaminants that 

had adsorbed onto the surface during

transport in air and not removing the

particles before they are analyzed. 

18000 Sn

16000

14000

T

a thickness of <3 nanometers. The thickness was determined by argon ion sputtering with a 

calibrated etch rate for different lengths of time until the carbon and oxygen peaks were

completely removed from the Auger spectrum. This was repeated at multiple locations on the

surface of the copper plate to assess the

variability and determine the maximum

contamination film thickness. The Auger results 

have provided the NIST researchers with the

feedback they needed to continue development

of important new technologies in the field of

microelectronics.

0

2000

4000

6000

8000

10

Counts

000

12000

200 300 400 500 600 700 800 900 1000

Energy (eV)

O Sn

Auger spectrum of contaminant particles 

on Sn-Cu film.

Recently, we have measured transition line 

shapes and strengths near a wavelength of 

940 nm and the temperature dependence of 

the water continuum absorption at a 

wavelength of 10 μm. 
147

11. Measurement Standards 

... anchoring the Nation’s measurement infrastructure and supporting mature and 

emerging technologies 

NIST provides a wide variety of measurement, standards, and data services and programs to 

help U.S. industry improve its international competitiveness, commercialize new technology, 

and achieve total quality in all facets of business operations. CSTL maintains and disseminates 

measurement standards for chemical, biomolecular, and chemical engineering measurements 

by developing Standard Reference Materials (SRM®s), Standard Reference Data (SRD), and 

by providing Calibration Services. CSTL partners with NIST Technology Services to help 

disseminate the standards to NIST customers. 

With a significant role in NIST’s Calibration 

Services, CSTL provides calibrations for 

volumetric test measures, pressure and 

vacuum gauges, thermometers, 

hydrometers, humidity measuring 

instruments, fluid flow meters and air 

speed instruments and optical filters. 

These calibrations help customers 

achieve the highest measurement quality 

and productivity. In addition, 

instrumentation manufacturers and other 

users rely on our calibration services to 

provide traceability to U.S. national 

measurement standards. In FY 2003, 

CSTL completed 1,501 calibrations with 

180 customers served. The Process 

Measurements Division and the Analytical Chemistry Division were responsible for the large 

majority of calibrations within CSTL in FY 2003. 

SRMs provide scientists and engineers in industrial and academic 

research with internationally accepted standards for critical technical 

decision-making. NIST pioneered, and continues to lead, in the 

development of certified reference materials used for quality assurance. 

Traditionally, SRMs have been the primary tools that NIST provides to 

the user community for achieving chemical measurement quality 

assurance and traceability to national standards. Currently, NIST 

catalogs nearly 1,440 SRMs; in 2003 NIST sold approximately 31,000 

SRM units to 

about 6,500 unique customers. 

Approximately 24,000 of the units sold 

were from the ≈ 700 different types of 

materials that are certified for chemical 

composition. CSTL provides technical 

leadership for most of the chemical and 

compositional standards produced by 

NIST. SRMs are used for three main 

purposes: to help develop accurate 

methods of analysis (reference 

methods); to calibrate measurement 

systems; and to assure the long-term 

adequacy and integrity of measurement 

Chemical Standards Development 

Examples of new reference materials in 

production from CSTL: 

Health - Human cardiac troponin complex, toxic 

elements in urine

Food - Ephedra standards, nutrients in peanut 

butter, and slurried spinach 

Environmental - Contaminants in fish tissue and 

marine sediment, atmospheric PM2.5, 

Chemicals - Low carbon steel, zeolites 

Commodities - Aluminum alloys, electronic 

scrap artifact Forensics/Homeland Defense - Smokeless gun 

powder, drugs of abuse, Y-chromosome DNA 

Physical Standards Development 

Examples of Physical Standards research 

activities in CSTL: 

Temperature – Primary acoustic thermometry, 

Methods and devices to disseminate ITS-90 

Pressure – Intrinsic pressure standards in the 

range of 0.3 MPa to 5 MPa 

Vacuum - Next-generation vacuum gauging 

technology 

Flow – New volumetric liquid flow standards 

reduces uncertainty by 10-fold 

Humidity - Rapid, low-cost methods for 

calibration of permeation tube generators 
148

quality assurance programs. NIST SRMs also legally constitute part of the National 

Measurement System infrastructure of the United States and, as such, are essential transfer 

mechanisms for national as well as international measurement traceability. 

To meet the growing need for traceable standards, CSTL pioneered the 

NIST Traceable Reference Materials (NTRM) program. An NTRM is a 

commercially produced reference material with a well-defined traceability 

linkage to existing NIST primary standards. The first NTRM program in gas-mixture reference 

materials was developed in 1992 for the compressed gas industry. The high availability of gasmixture reference materials as NTRMs has significantly 

aided vehicle emissions testing in the U.S. and has 

become the cornerstone of emissions trading in the 

fossil-fueled electric power industry. In 2003 137 gas 

SRMs were recertified for 14 Specialty gas companies. 

Convinced of the success of the NTRM program, CSTL 

is examining this model as well as other mechanisms to 

reach markets where a high demand for NIST traceability 

exists. 

One of these mechanisms by which CSTL meets the 

need for additional traceable standards is intrinsic 

standards development. Intrinsic standards can reduce 

the need for labor-intensive artifact standard production 

and repeated costly inter-laboratory measurement 

comparisons. A paradigm is envisioned in which 

appropriate SRMs are not supported in perpetuity, but 

are produced through enough cycles to establish the 

material as an intrinsic standard or transfer the continued 

production to the private sector with an acceptable traceability strategy. A specific step in this 

direction is the imminent publication of internationally determined band positions of dilute acidic 

holmium oxide solution as an intrinsic wavelength standard for calibration in spectrophotometry. 

In a related development, CSTL researchers have developed an algorithm to implement 

holmium oxide solution wavelength calibration and a proposed extended use of the material as 

a universal simultaneous standard for both wavelength and absorbance. Additional details are 

provided in the following highlight. 

Development and Dissemination of Intrinsic Standards for Chemical Measurements 

P.M. Chu, D.L. Duewer, M.L. Salit, and J.C. Travis 

Three separate efforts within the CSTL report 

significant progress in the development of intrinsic 

standards. (1) Samples of SRM 2034 (dilute holmium 

oxide solution sealed into fused silica cuvettes) were 

distributed to fifteen NMIs. Results from these 

laboratories, NIST/PL, and our own reference 

instruments will form the basis for an internationally 

supported intrinsic wavelength standard for UV/visible 

chemical spectrophotometry. (2) In collaboration with 

the NIST Physics Laboratory (NIST/PL), we have 

prepared the first of three manuscripts reporting improved spectral line positions for mercury 

and argon atomic lines in low-pressure discharges. These positions serve as secondary length 

A study conducted by RTI 

International documented the 

success of the gas-mixture NTRM 

program, estimating that the “net 

benefits” of the program projected 

through 2007 will be $50M to $63M 

with a social rate of return of 

about 225%. 

Intrinsic standards “based on 

well characterized laws of 

physics, fundamental constants 

of nature, or invariant properties 

of materials” (ANSI/NCSL Z540) 

have many uses in physical and 

chemical metrology. 

Intrinsic standards enable 

National Metrology Institutes 

(NMIs) to concentrate efforts on 

metrology rather than artifact 

production or calibration, and 

offer end-users of commercial 

standards ready access to 

traceability through data.
149

standards and support higher accuracy in the determination of atomic line positions in such 

analytical plasmas as the Inductively Coupled Plasma (ICP). (3) SRD 79 Quantitative Infrared 

Database is an on-going project providing qualityassured quantitative infrared absorption 

reference data to support open-path Fourier 

transform infrared (FT-IR) measurements, such 

as those described in EPA Method TO-16. New 

spectra based on gravimetrically prepared 

samples are added to the database as they are 

acquired. Comparisons with Pacific Northwest 

National Laboratories continue to play a key role 

in validating the data. 

The NIST Gravimetric Hygrometer: A Primary Humidity Standard 

C. W. Meyer, G.E. Scace, J.T. Hodges (836); 

J. Valencia (Guest Researcher, CICATA, Mexico) 

The NIST gravimetric hygrometer (GH)

hygrometer is a primary humidity standard 

that determines the amount of water in a 

gas sample by measuring the mass fraction 

of the water. This is accomplished by 

trapping the water in collection tubes 

containing desiccant; the collection tubes 

are weighed before and after trapping, and 

the mass difference is due to the trapped 

water. The mass of the dried gas sample is determined by volume measurements using 

interferometric measurements of piston displacement (see figure), in combination with gas 

density calculations. Performance optimization and uncertainty evaluation are accomplished 

through both control tests and measurement of the 

humidity from a well-characterized generator. 

The GH is now operational and its uncertainties are 

currently being evaluated and minimized. 

Comparisons with the NIST Low Frost Point 

Generator and 2-Pressure Generator have 

determined its present agreement with these 

generators to be within 0.4 % of water mass fraction. 

The comparisons have also shown the present dayto-day reproducibility of GH measurements to be within 0.3 %. Control tests have identified 

methods for further reduction of the measurement uncertainties. Specifically, tests have been 

Economy of scale encourages users to 

purchase the physical realization of an 

intrinsic standard (an artifact transfer 

standard) rather than constructing it 

themselves. Thus, the production of 

artifact standards devolves from the NMI 

to the commercial sector, which is better 

suited to production. 

NIST humidity calibrations are performed by 

generating samples of gas with well-characterized 

humidities, which are then supplied to customer 

hygrometers. To insure the highest quality 

calibrations, the performance of these generators 

should be periodically tested by measurement of 

the generated humidity using a primary humidity 

standard. The GH was built to fulfill this function. 

Prover tubes with pistons used for the 

determination of the sample volume. 

NIST has a primary working standard for 

humidity that can validate the performance 

of the generators used for calibrating 

customer hygrometers. This helps assure 

NIST customers that its humidity 

standards and calibrations are maintained 

at the levels needed by industry.
150

performed to determine the water collection capacity of the collection tubes and to accurately 

account for changes of gas mass in the collection tubes. Additional tests have been designed to 

determine the amount of residual water in the gas after it passes through the collection tubes. 

Elucidation of Systematic Effects in Primary pH Measurements 

K.W. Pratt (839) 

SRMs provide the primary benchmarks to establish the 

traceability of pH measurements. International 

recognition of these SRMs is supported by NIST 

participation in Key Comparisons (KC), administered by 

the Conseuil consultatif pour la quantité de matière 

(CCQM), which is described in more detail in the International Measurement Standards

section of this chapter. NIST performance in prior pH KCs has been sufficient to ensure 

international recognition of the pH SRM suite. However, the reproducibility of the NIST primary 

pH measurements in these KCs and in prior SRM certifications has been degraded by largerthan-expected Type A uncertainties (“scatter”) of the measurement replication in the primary pH 

measurements. The primary measurement yields the standard pH values, designated pH(S), of 

the given buffer. The Type A uncertainties in these pH(S) values have been larger than 

expected, based on analysis of the set of expected Type B (systematic) components of the 

overall uncertainty in the primary pH(S) measurement. This state of affairs suggested that an 

additional, uncontrolled factor, hitherto not considered in the uncertainty analysis, limited the 

reproducibility of primary pH metrology as realized at NIST. A fundamental study was initiated 

to elucidate this unknown factor. Transient mixed potential phenomena arise within Ag|AgCl 

electrodes, used in the primary measurement, following their transfer from the storage solution 

(0.01 mol kg-1 HCl) to the pH buffer being certified. These transient phenomena decay to a 

negligible level if the electrodes are stored in the given buffer solution for at least 12 h prior to 

performing the primary measurement. This pre-equilibration step has been incorporated into 

the NIST procedure for primary pH measurements. The results of the fundamental study into 

the transient behavior of Ag|AgCl reference electrodes provided new information regarding the 

systematic uncertainties and behavior of Ag|AgCl electrodes 

in primary pH measurements. These fundamental studies will 

be continued, yielding explanations of transient phenomena in 

Ag|AgCl electrodes based on modern electrochemical theory. 

The quality of primary pH measurements obtained at NIST 

now equals or exceeds that of other NMIs that currently 

perform similar measurements. Based on these pH SRM 

certifications, it is expected that the NIST performance in 

future CCQM pH KCs, as evaluated by the Type A uncertainties that are attained, will surpass 

the corresponding “best” level attained by the set of NMIs participating in the given CCQM pH 

KC. 

CSTL supports the certification 

of seven pH SRM buffers 

covering a range of roughly pH 

1.7 to 12.4. 

Changes in the NIST 

protocol for primary 

measurements of pH(S) have 

already yielded significant 

improvements in the 

uncertainty obtained in 

certifications of pH SRMs. 
151

Selected Chemical and Physical Standards Activities in FY 2003 

(Described in more detail elsewhere in this report) 

Automotive and Aerospace 

New Gas Standards for Calibrating Instrumentation used for Measuring Emissions from 

Next Generation Low Emission Vehicles 

W.J. Thorn III and W.D. Dorko (839) 

Pharmaceuticals and Biomanufacturing 

Use of SRM Based Column Performance Tests to Guide LC Column Selection in the 

Pharmaceutical Industry

L.C. Sander and S.A. Wise (839)

New Glass Filter Reference Materials for Validating the Performance of UV/visible 

Spectrophotometers in the High Absorbance Range 

J.C. Travis, M.V. Smith, and G.W. Kramer (839) 

Chemical and Allied Products 

Zeolite Reference Materials: Chemical Properties, Structural Parameters, and Particle 

Size Distribution 

S. Turner, R.A. Fletcher, E.S. Windsor, and R.R. Cavanagh (837); J.R. Sieber, T.W. Vetter, 

R.L. Zeisler, and D.A. Becker (839); B.H. Toby and J. Kelly (MSEL); S.D. Leigh (ITL); M. 

Davis (Caltech); S. Yang and A. Navrotsky (Univ. California at Davis) 

International Standards for Refrigerant Properties 

M.O. McLinden and E.W. Lemmon (838)

Measurements, Modeling, and Data for Pressure-Driven Membrane Separations 

C. Muzny, H. Sun, and J. Moon (838); J. Cho (U. of Colorado); M. Chapman (U.S. Bureau 

of Reclamation); and J. Ranville (Colorado School of Mines) 

Energy and Environmental Technologies 

Natural Gas Flow: North American Laboratory Comparison Project 

P. I. Espina (836) and W. F. Guthrie (ITL) 

Influence of Oxygen on the Shelf Life of Coal SRMs 

R. Zeisler (839) and W.D. James (Texas A&M) 

SRMs for Air Particulate Matter Less Than 2.5 μm (PM2.5) 

R. Zeisler, J.R. Kucklick, B.J. Porter, D.L. Poster, M.M Schantz, R.O. Spatz, and S.A. Wise 

(839) 

Developing Methods for the Determination of Past-Use and Current-Use Persistent 

Organic Pollutants in Standard Reference Materials 

J.R. Kucklick, M.M. Schantz, S.A. VanderPol, K.J. Tuerk, and S.A. Wise (839) 

SRMs for Contaminants in Marine Sediment and Aquatic Species Tissue 

S.A. Wise, S.J. Christopher, R. Demiralp Oflaz, R.R. Greenberg, J.R. Kucklick, S.E. Long, 

E.A. Mackey, B.J. Porter, D.L. Poster, M.M. Schantz, and R. Zeisler (839) 
152

International Infrastructure for Traceability in Atmospheric Ozone Measurements 

P.M. Chu and J.E. Norris (839) 

Development and Measurement of Heavy Hydrocarbon (C6-C16) Gas Standards 

G.C. Rhoderick (839) 

Food and Nutrition 

Development of Analytical Methods and SRMs for Chemical Characterization of Botanical 

Dietary Supplements 

S.A. Wise, T.A. Butler, S.E. Long, M.C. Mildner, E.A. Mackey, K.E. Murphy, K.W. Phinney, 

B.J. Porter, L.C. Sander, M.B. Satterfield, K.E. Sharpless, L.J. Wood, and L.L. Yu (839); 

T. Ihara (National Metrology Institute of Japan, AIST). 

Certification of SRM 1946 Lake Superior Fish Tissue – Frozen Fish Tissue for 

Environmental Contaminants and Food Constituent Measurements 

S.A. Wise, S.J. Christopher, J.R. Kucklick, S.E. Long, E.A. Mackey, C.S. Phinney, B.J. 

Porter, R.S. Pugh, D.L. Poster, M. Rearick, KE. Sharpless, and M.M. Schantz (839) 

A Complementary Suite of Food-Matrix SRMs to Support Industry Compliance with 

Nutritional Labeling Requirements 

K.E. Sharpless, J. Brown-Thomas, C.S. Phinney, B.J. Porter, and L.J. Wood (839) 

Keeping Tin Cans Lead-Free 

J.R. Sieber, K.E. Murphy, and S.E. Long (839) 

Forensics and Homeland Security 

Certification of NIST Reference Material Supporting Improvised Explosive Device 

Measurements 

W.A. MacCrehan and M. Bedner (839) 

Standards for Detection of Biological Threat Agents 

K. D. Cole, L. Wang, A. Gaigalas, and D. Hancock (831) 

Health and Medical Technologies 

Standards Requirements for Systems Biology Approaches to Health Care: Mitochondrial 

Proteomics 

Gregory B. Vásquez and Peter E. Barker (831); Steven Zullo (ATP/NIST) 

Development of Reference Methods and Reference Materials for Clinical Diagnostic 

Markers 

M.J. Welch, D.M. Bunk, M. Satterfield, L.T. Sniegoski, and S.S-C. Tai (839) 

Development of SRM 2921, Human Cardiac Troponin Complex: A Primary Calibrator for 

Assays Used to Diagnose Heart Attacks 

D.M. Bunk and M.J. Welch (839) 

Development of Reference Methods and Participation in International Round-Robins for 

the Determination of Thyroid Markers 

S. S-C. Tai, D.M. Bunk, and M.J. Welch (839) 
153

Reference Methods and SRMs for Toxic Species in Body Fluids 

S.E. Long, R.D. Vocke, L.L. Yu, K.E. Murphy, T.A. Butler, and E.A. Mackey (839) 

Standards for Fluorescence Microarray Analyses 

G.W. Kramer (839); A.G. Gaigalas (831); P.C. DeRose (839) 

Industrial and Analytical Instruments and Services 

Standards for Raman Spectroscopy 

W.S. Hurst, J.E. Maslar (836); S.J. Choquette (839); and E.S. Etz (837) 

Fluorescence Standards Suite for Spectral Correction of Fluorometers 

P.C. DeRose, D.H. Blackburn, and G.W. Kramer (839) 

Improved Vacuum Transfer Standards – Ionization Gauges 

P.J. Abbott (836); P. Mohan (NPL India) 

Development of Critical Benchmark Standards and Methods for the Metals Industry 

J.R. Sieber, A.F. Marlow, M.R. Winchester, L.L. Yu, T.A. Butler (839); R.M. Lindstrom 

(837); M.P. Cronise, and C.N. Fales (TS) 

Elucidation of Systematic Effects in Primary pH Measurements 

K.W. Pratt (839) 

Microelectronics 

Electron Probe Characterization of KNbO3 and NaNbO3 Single Crystals for use as 

Quantitative Microanalysis Reference Materials 

R. B. Marinenko (837), Z. Samardžija, S. Bernik, B. Malič and M. Čeh, (Jožef Stefan 

Institute, Slovenia) 

Development of Phosphorus Ion Implant in Silicon Depth Profile Standard 

D. Simons (837), R. Paul (839), W. Guthrie and J. Lu (ITL) 
154

http://kcdb.bipm.org/BIPM-KCDB/ 

The objectives of the MRA are: 

• To establish the degree of equivalence of national measurement standards maintained by 

NMIs; 

• To provide for the mutual recognition of calibration and measurement certificates (CMCs) 

issued by NMIs; and 

• Thereby to provide governments and other parties with a secure technical foundation for the 

wider agreements related to international trade, commerce, and regulatory affairs. 

International Measurement Standards 

Increased requirements for quality systems 

documentation for trade and effective decisionmaking regarding the health and safety of the U.S. 

population have increased the need for 

demonstrating “traceability to NIST” and establishing 

a more formal means for documenting measurement 

comparability with standards laboratories of other 

nations and/or regions. 

NIST is the U.S. National Metrology 

Institute (NMI) and the agency of the 

U.S. Government responsible for U.S. 

efforts under the Treaty of the Metre. 

The Committee International des Poids 

et Mesures (CIPM), and its various 

consultative committees, organizes 

comparisons of national measurement 

standards. After review, the results of 

these comparisons are recorded in the 

BIPM database (Appendix B). In 

addition, coordination of similar efforts 

with Regional Metrology Organizations 

(RMOs) extend the comparison efforts 

to as many participants as practicable. 

In October 1999, NIST signed the 

CIPM Mutual Recognition 

Arrangement (MRA). 

Since 1999 CSTL has systematically compared U.S. national measurement 

standards to establish degrees of equivalence of U.S. national measurement standard with 

those of other NMIs. These international activities add value to NIST standards and services, 

particularly for our customers involved in international trade. These MRA-related activities 

guarantee recognition of U.S. standards by U.S. trading partners. 

For implementation of this MRA, the signatory NMIs agreed to: 

1. Declare and document their calibration and measurement capabilities (CMCs) 

 [Appendix C] (http://kcdb.bipm.org/AppendixC/default.asp)

2. Participate in relevant international comparisons to support their CMCs 

 [Appendix B] (http://kcdb.bipm.fr/BIPM/KCDB/; http://icdb.nist.gov )

3. Implement and document the existence of a system for assuring the quality of the 

measurement services provided. 
155

Regional Metrology Organizations (RMOs) play 

an important role in the MRA. They have the 

responsibility for carrying out key comparisons 

within their regions. They also carry out 

supplementary comparisons and other actions 

to support mutual confidence in the validity of 

calibration and measurement certificates 

through the Joint Committee of the RMOs and 

the BIPM (JCRB). They are also responsible 

for the entries into the BIPM key comparisons 

database for the calibration and measurement capabilities of their member NMIs. 

Systema Interamericano de Metrologia (SIM) is the RMO that includes the United 

States. The metrology organizations that make up SIM are known as: NORAMET 

(North America), CAMET (Central America), CARIMET (Carribean), ANDIMET 

(Andes Region), and SURAMET (South America). Regional cooperation leads to 

a wider harmonization of measurements and standards, facilitates free flow of 

trade, and is a necessary first step to globalization. NIST and National Research Council 

(NRC) Canada link SIM countries to the BIPM, and as such, provide greater opportunities for 

international trade and commerce. CSTL staff provides leadership for SIM by chairing the 

Chemical Metrology Working Group of SIM, and by serving as the U.S. respresentative to the 

JCRB, in order to assure the effective, fair, and metrologically sound implementation of the 

MRA. 

CIPM Consultative Committees (CCs) 

The CIPM has set up a number of Consultative Committees, which bring together the world's 

experts in their specified fields as advisers on scientific and technical matters. Among the tasks 

of these Committees are the detailed consideration of advances in physics that directly 

influence metrology, the preparation of Recommendations 

for discussion at the CIPM, the identification, planning and 

execution of key comparisons of national measurement 

standards, and the provision of advice to the CIPM on the 

scientific work in the laboratories of the BIPM. 

CIPM Consultative Committees for Temperature (CCT) 

and Mass (CCM) 

The Process Measurements Division has significant 

participation in the CC’s for Temperature (CCT) and Mass 

(CCM). We have lead or participated in many Key 

Comparisons in the past several years. In thermometry, 

these efforts have resulted in establishing equivalence 

levels over the ITS-90 range from 14 K to 1235 K. Key 

Comparisons organized by the CCM include Division 

activities in both the pressure and vacuum and the flow 

project areas. In the pressure and vacuum project the 

Division leads or participates in Key Comparisons that 

cover the pressure range 3x10-6 Pa to 500 MPa. NIST has 

piloted three CCM Key Comparisons in the last several 

years, completing two in FY02 that demonstrated general 

equivalence among the participants, revealed no 

systematic bias between alternative realizations of the 

In order to most effectively address the 

unique needs of all 32 countries within SIM, 

CSTL has initially focused the SIM program 

on training and capability assessment 

rather than participation in MRA-driven 

KCs. During the past three years, 16 

intercomparison exercises were carried out 

to assess the proficiency of SIM NMIs 

and/or their designated laboratories. Four 

additional exercises are planned for 2004. 

GLOSSARY 

BIPM Bureau International des 

Poids et Mesures 

CIPM Committee International 

des Poids et Mesures 

CC Consultative Committees

CCM Consultative Committees 

for Mass 

CCQM Consultative Committee 

on the Quantity of 

Material 

CCT Consultative Committees 

for Temperature 

JCRB Joint Committee of the 

RMOs and BIPM 

KC Key Comparisons 

KCRV Key Comparison 

Reference Values 

MRA Mutual Recognition 

Arrangement/Agreement

RMO Regional Metrology 

Organizations 

SIM Systema Interamericano 

de Metrologia 
156

Pascal, were the only CCM Key Comparisons completed on schedule, and set the standard for 

the manner in which Key Comparisons should be conducted. In addition, CSTL researcher G. E. 

Mattingly continues to chair the CCM’s Working Group for Fluid Flow (WGFF). Through FY 03, 

this working group has progressed applying its strategy to produce Key Comparisons (KCs) in 

seven different flow measurement areas: water, 

hydrocarbon liquids, low-pressure air, highpressure and high-flow natural gas, high pressure 

nitrogen or air, air speed, and liquid volume. In 

each of the seven flow areas, different NMIs have 

accepted the responsibility to produce transfer 

standards and the test procedures needed to 

compare the respective flow standards of the 

participating NMIs. One of these - the Korea 

Research Institute of Standards and Science (KRISS) has completed this work in water flow and 

with approvals from the CCM, has officially started the working 

group’s first KC. This KRISS program is expected to set 

precedents that should advance the state-of-the-art in 

conducting all future flow laboratory comparisons. The Working Group for Fluid Flow chair 

continues to work closely with the NIST Statistical Engineering Division to develop sound, 

statistically based test procedure designs and supporting analysis methods applicable to all 

KCs, and to assess the metrological linkage techniques that will be required to connect the 

results of the KCs to the associated Regional Metrology Organization (RMO) tests that will 

follow the KCs to metrologically link all of the participating NMIs. 

NIST will pilot the KC for the low-pressure gas flow. Recent advances in U.S. standards and 

procedures (described elsewhere in this report - see citation below) are expected to materially 

improve the performance-level of this KC. Characterization of the transfer package will begin in 

FY04. 

Natural Gas Flow: North American Laboratory Comparison Project 

P. I. Espina and W. F. Guthrie (898) 

Details provided in the Energy and Environmental Technologies section.

Consultative Committee on the Quantity of Material (CCQM)

The Consultative Committee on the Quantity of Material (CCQM) has seven working groups: (1) 

Gas Analysis, (2) Organic Analysis, (3) Inorganic Analysis, (4) Electrochemistry (5) 

Biometrology, (6) Surface Analysis and, (7) Key Comparisons. These working groups are 

responsible for selecting and overseeing the operation of key comparisons that address 

chemical measurement-related issues important for international trade, environmental, health, 

and safety-related decision making. CSTL staff are active in all seven and has provided formal 

leadership for the Organic Analysis, Biometrology 

and Key Comparisons Working Groups. During the 

past five years, approximately 96 comparison studies 

have or are being conducted under the auspices of 

the CCQM. CSTL’s Analytical Chemistry Division 

(ACD) has participated in 75 of these, serving as 

Coordinating Laboratory in 31. Over 25 additional 

studies are planned over the next two years and 

NIST has already committed to coordinate at least 

seven of these. In FY03, the comparisons in which CSTL participated were for the determination 

of a variety of organic and inorganic components in matrices such as gaseous mixtures, 

sediments, food, water, fuel, and serum. 

NIST’s Quality Manual for Chemical 

Measurements summarizes and 

formalizes policies and approaches 

for addressing quality-related 

issues concerning the services that 

it provides is being updated to 

assure appropriate compliance with 

ISO/EC 17025 and ISO Guide 34. 

NIST’s Quality Manuals for calibrations 

are basically equivalent to ISO 17025 

Requirements for the Competence of 

Testing and Calibration Laboratories and 

its calibration services only needed 

relatively minor upgrades to comply with 

the NIST-wide objectives of ISO17025 

equivalency by December 2003. 
157

Participation in relevant key comparisons (documented in MRA Appendix B) serve to 

benchmark the claims made in the Calibrations and Measurements Capabilities (documented in 

MRA Appendix C). CSTL’s Analytical Chemistry Division has led the critical review of CMC data 

for Appendix C in the BIPM Database. SIM has approximately 1140 CMC entries scheduled for 

inclusion in the BIPM database. By the beginning of 2004, the BIPM database included 

approximately 3000 CMCs for chemistry, with about 1000 from NIST. 

Selected CCQM KCs Completed in FY 2003 

Results of CCQM-K25 Key Comparison for Polychlorinated Biphenyl Congeners in 

Sediment 

M.M. Schantz and S.A. Wise (839) 

Polychlorinated biphenyls (PCBs) consist of 

209 possible congeners depending on the 

substitution of the chlorine atoms around the 

biphenyl molecule. PCBs have been widely 

used as industrial fluids, flame retardants, 

diluents, hydraulic fluids, and dielectric fluids 

for capacitors and transformers. As a class of 

compounds, they are environmentally stable 

and tend to bioaccumulate. Of the 209 possible congeners, approximately 150 congeners have 

been reported in the environment. A CCQM key comparison for PCB congeners in sediment 

was conducted among nine laboratories, with NIST serving as the coordinating laboratory. All 

but one of the participant laboratories used gas chromatography/mass spectrometry (GC/MS) 

with carbon-13 labeled PCB congeners as internal standards/surrogates (isotope dilution (ID)-

GC/MS). 

Five PCB congeners were chosen: PCB 28, PCB 101, PCB 105, PCB 153, and PCB 170. PCB 

28 (2,4,4’-trichlorobiphenyl) is volatile and under certain conditions coelutes with PCB 31 

(2,4”,5-Trichlorobiphenyl). PCB 101 (2,2’,4,5,5’-pentachlorobiphenyl) has the potential coelution 

with a minor congener, PCB 90 (2,2’,3,4’,5-pentachlorobiphenyl). PCB 153 (2,2’,4,4’,5,5’-

hexachlorobiphenyl) is typically one of the most abundant congeners and potentially coelutes 

with PCB 132 (2,2’,3,3’,4,6’-hexachlorobiphenyl). PCB 105 (2,3,3’,4,4’-pentachlorobiphenyl) is a 

congener with a lower concentration than 

the other congeners selected and which 

may change elution order with PCB 132 

depending on the analytical conditions. 

Finally, PCB 170 (2,2’,3,3’,4,4’,5-

heptachlorobiphenyl) is one of the less 

volatile congeners, is typically found at 

lower concentrations, and can potentially 

coelute with PCB 190 (2,3,3’,4,4’,5,6-

heptachlorobiphenyl). The ability of a 

laboratory to measure these five 

congeners should indicate their ability to 

measure the suite of 150 PCB congeners 

found in sediments. 

The uncertainties of the key comparison reference values (KCRVs) ranged from 2 % to 5 % 

indicating excellent agreement among the participants. The agreement among laboratories and 

This Key Comparison demonstrated a 

high level of equivalence in capabilities 

of the participating national metrology 

institutes to successfully measure PCB 

congeners 28, 101, 105, 153, and 170 in 

a moderately contaminated sediment 

using ID-GC/MS.

KCRV: 10.55 ng/g (dry basis) + 0.45 ng/g (dry basis)

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

BAM IRMM LGC NARL NIST NMIJ NRC

NMI

Diff. from KCRV (ng/g) 

Diff. from KCRV (%) 

-19

-9.5

0.0 

 9.5

CCQM K25 PCB 105 Equivalence 
158

CCQM K14: Calcium in Serum 

Results for 8 NMIs (NIST shown in red) 

comparison to the KCRV is shown in the figure for PCB 105. Similar degrees of equivalence 

were demonstrated for the other four congeners. 

Results From the CCQM-K14 Key Comparison for Calcium in Serum 

S.E. Long and K.E. Murphy (839) 

Under the auspices of the CCQM 

Inorganic Analytical Working Group, the 

CCQM-K14 Key Comparison addressed 

the need for better measurement 

methodology for routine clinical 

measurements of total calcium in blood 

serum. It is evident that the current 

measurement system is unsatisfactory 

from a clinical diagnostic standpoint, and 

the resulting impact on health-care costs 

may be considerable. 

Following the successful participation by the 

Analytical Chemistry Division in the pilot study during 

FY01, calcium was determined in an un-modified 

frozen human serum, which was distributed by 

European Commission’s Institute for Reference 

Materials and Measurements (IRMM), the 

coordinating institute for this study. Eight other 

national metrology institutes also participated. 

Calcium was determined using a method employing isotope dilution “cold-plasma” inductively 

coupled plasma – mass spectrometry (ICP-MS). The result ((2.2395 ± 0.0115) mmol.kg-1) was 

in excellent agreement with the other laboratories and also the proposed value for the key 

comparison reference value, which was (2.240 ± 0.008) mmol.kg-1. The analytical data from the 

key comparison are shown below. It is 

interesting to note that there has been a shift 

from using thermal ionization mass 

spectrometry (TIMS) for making such 

measurements. The shift to ICP-MS has been 

made possible by the advent of systems for 

reducing or eliminating spectral interferences. 

Of the nine participants in the study, only two 

used TIMS. The remainder used a 

combination of high-resolution sector field, 

collision cell and cold-plasma ICP-MS, while 

one laboratory used ICP optical emission 

spectrometry. 

The Analytical Chemistry Division achieved 

excellent agreement with other national 

metrology institutes in a CCQM Key 

Comparison of calcium in blood serum (K14). 

The calcium determination was by isotope 

dilution inductively coupled plasma – mass 

spectrometry. Measurements were nearly 

identical to the key comparison reference 

value chosen for this Key Comparison. 

We have demonstrated the veracity of 

the analytical measurement system 

employed here in this Key 

Comparison for this calcium in blood 

serum. Such comparisons provide 

documented performance for the NIST 

quality system. 
159

Results from International Comparisons for Selected Components in Natural Gas 

G.C. Rhoderick and F.R. Guenther 

NIST participated in the CCQM-K16 Natural Gas Key Comparison, for which the final report was 

released in October of 2002. The study evaluated the degree of equivalence among national 

metrology institutes (NMIs) for the given 

measurand in natural gas. Unlike past 

comparisons, this natural gas included heavier 

hydrocarbons and was designed to emulate real 

natural gas at both high and low calorific value. 

NIST completed the analysis of the low calorific 

value natural gas standard in FY03 and released 

the report to the coordinating laboratory. The 

table below lists the NIST results of the 

comparison. 

* Expanded uncertainty U = kuc, with the combined standard uncertainty, uc, determined from experiment 

and a coverage factor k = 2. The true value for the analyte amount-of-substance fraction is asserted to lie 

in the interval defined by the NIST value ± U with a level of confidence of approximately 95 %. 

A similar study was organized by NIST as a pilot for the SIM countries. Since this was a pilot 

study, the results will not be made available publicly. All the participating labs submitted 

methane values that were within ± 1.0 % of the certified concentration. However, the agreement 

among labs for all of the remaining 

compounds was poor. One lab was 

consistently within ± 1.5 % of the certified 

values with the exception of carbon dioxide 

(4.6 %) and n-butane (1.7 %). A SIM pilot 

study on automobile emission gases, piloted 

by NIST, will be completed by March 2004. A 

new SIM pilot study on industrial gas 

emission (sulfur dioxide) piloted by NIST will be initiated in late FY2004. 

Agreement between NIST value and Preparation Value (given as % mol mol-1) 

Compound Reference 

Value 

Reference 

Value 

Uncertainty*

NIST 

Value 

NIST 

Value 

Uncertainty*

Absolute 

Difference 

∆ x 

Relative 

Difference

∆ x/x

Methane 81.6826 0.0027 81.77 0.46 0.087 0.11 %

Nitrogen 12.1205 0.0007 12.11 0.16 -0.010 -0.09 %

CO2 4.0446 0.0005 4.055 0.026 0.010 0.26 %

Helium 0.50813 0.00016 0.5158 0.0026 0.0077 1.51 %

Ethane 0.7489 0.0002 0.7493 0.0046 0.0004 0.05 %

Propane 0.29144 0.00010 0.2905 0.0026 -0.0009 -0.32 %

iso-Butane 0.20165 0.00019 0.2034 0.0019 0.0018 0.87 %

n-Butane 0.20260 0.00007 0.2024 0.0018 -0.0002 -0.10 %

iso-Pentane 0.04861 0.00011 0.0483 0.0006 -0.00031 -0.64 %

n-Pentane 0.04982 0.00011 0.0500 0.0007 0.00018 0.36 %

neo-Pentane 0.04972 0.00006 0.0495 0.0007 -0.0002 -0.44 %

n-Hexane 0.05061 0.00011 0.0502 0.0008 -0.0004 -0.81 %

The CCQM-K16 comparisons were designed 

to evaluate the capabilities of NMIs to certify 

natural gas standards. They are intended to 

provide evidence for calibration and 

measurement capabilities listed by each 

institute in the BIPM Mutual Recognition 

Arrangement Database. 

NIST’s results show excellent 

agreement with the gravimetric key 

comparison reference values. A pilot 

study similar to the key comparison 

was organized by NIST for the SIM 

countries. Results of this pilot study 

on natural reveal much larger 

deviations than those of the CCQM 

key comparison. 
160

Joint Committee on Traceability in Laboratory Medicine (JCTLM) 

Under the auspices of BIPM, the Joint 

Committee on Traceability in Laboratory 

Medicine (JCTLM) was created in June 2002 to 

provide a worldwide platform for promoting and 

guiding the internationally recognized and 

accepted equivalence of measurements in laboratory medicine and the traceability of these 

measurements to appropriate standards. The committee is charged with facilitating the 

identification of the “higher-order” reference measurement procedures and certified reference 

materials that are currently available which provide the traceability of values assigned to 

calibrators and control materials for in vitro diagnostic devices (IVD). 

At present, neither reference materials nor reference 

methods are available for more than 300 different 

chemical and biochemical species that are measured 

in medical laboratories using IVDs. Reference 

methods and/or materials exist for only about 30 

worldwide. The US IVD industry has asked NIST to 

work with our counterparts in Europe and the AsiaPacific to provide the reference materials and 

methods of “higher order” that are urgently needed to 

comply with the requirements of the EU IVD 

Directive. 

To facilitate the identification of “higher order” reference methods and reference materials that 

are currently available, the Joint Committee on Traceability in Laboratory Medicine (JCTLM) 

was created at a meeting held at the BIPM in early June 2002. Three meetings were held this 

year to review nominations for more than 80 Reference Methods and 435 Reference Materials 

in eight analyte categories.

Based on this work and input from medical 

professionals and IVD manufacturers, JCTLM 

Working Group I (Reference Materials and 

Reference Procedures) will also establish a 

list of highest priority needs for new reference 

procedures and reference materials. NIST, 

EU and AP Laboratories have also begun a 

dialogue to share the workload in developing 

and maintaining the more than 100 standards 

that are needed for compliance by the IVD industry with the EU IVDD. NIST has been asked to 

provide about 40 % of the standards based on our current capabilities and U.S. market share. 

Staff in CSTL’s Analytical Chemistry Division and Biotechnology Divisions are developing the 

“higher-order” reference materials and methods for the US IVD industry to comply with the 

requirements of the EU IVD Directive. These include the following health status markers: 

One requirement of the recently 

enacted European Directive 

98/79/EC on in vitro diagnostic 

medical devices is that “the 

traceability of values assigned to 

calibrators and control materials ... 

must be assured through available 

reference measurement 

procedures and/or reference 

materials of higher order.” 

The JCTLM created two working groups: 

I. Reference Materials and Reference 

Procedures 

II. Reference Laboratory Networks 

JCTLM Working Group I chaired by NIST and 

IRMM is charged with establishing a process 

for identifying, reviewing against agreed 

upon criteria, and publishing a List of “higher 

order” Certified Reference Materials and 

Reference Measurement Procedures required 

for IVD industry compliance with the EC IVD 

Directive. 
161

In 2003, work was completed for standards for Toxic Metals in Urine, Cardiac Troponin-I and 

Electrolytes in Human Serum. Work will continue on the high priority list presented above. 

SRMs will be completed for Homocystein, Folates, Thyroid Markers in 2004. Work will also 

begin on standards for gene expression including RNA standards and a fluorescence standard 

for microarray scanning devices. JCTLM Review Teams for Blood gases, Blood Groupings, 

Microbial Serology, Non-Electrolyte metals and Vitamins will be established for providing lists of 

“higher order” methods and materials by December 2004. 

Strategic Bilateral Collaborations and Intercomparisons with NMIs 

W.E. May, F.R. Guenther, S.A. Wise, and R.R. Greenberg (839) 

In addition to these global and regional activities, we are also establishing a limited 

number of strategic bilateral collaborations and intercomparisons with NMIs around 

the world. For example, our collaboration with the Netherlands Measurement 

Institute (NMi) for determining the equivalence of primary gas standards has 

resulted in a formal “Declaration of Equivalence” that is 

recognized by the U.S. EPA and 

European environmental regulatory 

bodies as documenting the equivalence 

between seven NIST and NMi primary 

gas mixture suites. Our formal 

agreement with NRC-Canada (via the NAFTA Treaty) for 

cooperation in marine environmental studies has fostered 

collaborations between the U.S. and Canada in the 

certification of a several certified reference materials 

important for trade and environmental decision-making. 

We have recently signed a Cooperative Arrangement with 

NIMC (AIST Japan) for collaborative efforts in the area of 

pure volatile organic compound standards. These highly pure reference compounds will serve 

as primary references for many of our gas mixture SRMs and our quantitative reference 

database (SRD-79). Several additional strategic 

bilateral arrangements with other National 

Metrology Institutes /Standards laboratories are 

being discussed. 

Health Status Marker Disease State

• Cardiac Troponin-I Heart attack, damage 

• Cadmium and MercuryToxic metal poisoning 

• Folates Neural tube defects 

• HER-2 Neu Breast cancer 

• Homocysteine Heart disease risk 

• Glycated Hemoglobin Diabetes status 

• Prostate-Specific Antigen Prostate cancer 

• Triiodothyronine and TSH Thyroid function 

• Trinucleotide Repeat Mental retardation 

• Speciated Iron Hemochromatosis, anemia 

CSTL’s Analytical Chemistry 

Division continues to work with 

CITAC (Cooperation in 

International Traceability in 

Analytical Chemistry) to 

establish practical, yet 

metrologically sound, vertical 

traceability links between the 

NMIs and chemical testing labs 

in the various countries and 

regions around the world. 
162
12. Data and Informatics 

... assuring that U.S. Industry has access to accurate and reliable data 

 and predictive models 

One of CSTL’s goals is to assure that U.S. industry has access to 

accurate and reliable data and predictive models to

determine the chemical and physical properties of

materials and processes. CSTL’s data and informatics

activities impact all industry sectors from biotechnology

and microelectronics to energy and instrument manufacturers. Versatile interactive databases 

provide easy access to high quality NIST data. Many databases are now available via the World 

Wide Web. The NIST Standard Reference Database (SRD)

series has grown to over 50 electronic databases in chemistry, 

physics, materials, building and fire research, software 

recognition, and electronics. Through this program CSTL 

provides SRDs for Analytical Chemistry, Atomic and Molecular

Physics, Biotechnology, Chemical and Crystal Structure,

Chemical Kinetics, Industrial Fluids and Chemical Engineering, 

Materials Properties, Surface Data, and Thermodynamics and 

Thermochemistry. A few of the highlights in the area of Data and

Informatics are described below, and a full listing of activities,

with references to the appropriate program section, is also

provided. 

CSTL continues to be a 

prominent source of SRD 

products at NIST. In FY 

2003, 4,225 SRD units from 

CSTL were sold by NIST. 

Among these, 734 units 

were sold directly to 

customers and 3,491 units 

were sold through 

distributors. 

In a number of data areas, including structural biology, thermodynamics, and kinetics, NIST 

researchers are working with other scientists and organizations to establish data standards and 

more rapid methods of data entry. One example is the Thermodynamics Research Center

(additional TRC details are provided in this section), which is working with several journals to 

have the thermodynamic data from accepted articles, go directly into the TRC database entry 

system through an electronic process. This assures that customers have the most up-to-date and

complete information possible. The creation of data transfer and traceability standards is another 

key area of development. These standards remove barriers to the sharing of information and

allow researchers to analyze results and collaborate in new ways. Another key concept is the

establishment of the pedigree of data, in which enough information is retained to easily trace 

results and assign uncertainties to measured values, thus answering the vital question: “How

good is that number?”. 

Usage of the WebBook has 

continued to grow at a rate of up to 

20% per year, currently over

650,000 distinct IP addresses 

access the WebBook per year. 

http://webbook.nist.gov/chemistry

The NIST Chemistry WebBook, remains one of the most used resources for chemical and 

physical property data. The numbers of users, between 10000 and 20000 per week, and the 

variety of users, in industry, government and academia is a clear indication of the success of the 

WebBook. The fraction of returning users, typically between 45 to 55%, is a 

good indication that the user community feels that the resource is valuable.

During 2003, the eighth edition of the NIST Chemistry WebBook was

released. More effort has gone into acquiring data and a major donation of

infrared spectral data from the Dow Chemical Company was received this year. In addition, the 

work on the IUPAC/NIST Chemical Identifier (INChI) has 

continued. The work on the INChI, along with work on 

data-exchange standards for chemical data is an

essential part of making the WebBook a leading part of

the next generation of electronic chemical information.

In addition to an enhanced profile of prediction tools, a

major increase in the infrared spectra was made with the

release of the digitized Coblentz Society infrared spectral

163
data. This data had only been available in printed form and the release of the data in fully 

digitized electronic form represents a major addition to the resources of the WebBook. 

The WebBook is also a tool to aid future evaluation projects both at NIST and in collaboration

with outside organizations. It is difficult to overstate the possible impact of the ongoing work on

developing standard protocols for transmission of chemical data. The need for such standards

has only grown as the use of the Internet in electronic commerce has grown. This need has 

been acknowledged by the large number of commercial as well as governmental entities 

participating in the IUPAC and ASTM committee work. 

CSTL also engages the broader community through 

conferences and seminar activities. CSTL’s Physical 

and Chemical Properties Division organized and cosponsored The 15th Symposium on Thermophysical

Properties held in Boulder, CO from June 22 to 27, 

2003. The community involved in thermophysical 

properties represents a diverse group of those involve

in their measurement, prediction, simulation, and 

application. The researchers and practitioners in the

field comprise physicists, chemists, biologists,

materials scientists, chemical engineers, mechanical 

engineers and others who may seldom have an opportunity to learn of the advances and 

impediments faced by their colleagues. 

The Symposium on Thermophysical 

Properties is a well-established series 

held roughly every three years since 

1959, and is recognized as one of the 

premier international conferences 

associated with the theoretical, 

experimental, simulation, and applied 

aspects of the thermophysical 

properties of gases, liquids, and 

solids, including biological systems. 

The conference attracted about 450 participants from more than 40 countries, 

with about half from outside the United States. The authorship of more than 

660 technical contributions – oral presentations, posters, and software

exhibitions – comprised more than 1500 individuals from 49 countries. The

conference consisted of 117 sessions including two plenary sessions, and

was organized by CSTL staff and other members of the international 

community of experts. There were specialized and more general focus areas 

devoted to properties of various classes of materials; properties for process 

design and environmental applications; techniques, including molecule 

simulation and novel experiments; phenomena; and tools for specialized 

thermophysical property problems. Planning is underway for the 16th Symposium, which will be 

held in 2006 in conjunction with the International Conference of Chemical Thermodynamics. 

Selected Data Activities for FY 2003 

Thermodynamics Research Center (TRC) Comprehensive Program on Critical Data 

Evaluation 

M. Frenkel, R. D. Chirico, G. R. Hardin, R. A. Stevenson (838); 

 Q. Dong, X. Yan, R. C. Wilhoit, X. Hong (Contractors); V. V. Diky, (Guest Researcher) 

The extraordinary growth of available data 

requires efficient new methods and

computer tools for collection, critical 

evaluation, mining, quality control, 

management, and dissemination of 

thermophysical, thermochemical, and

transport property data. The TRC concept

of “dynamic” data evaluation requires 

large electronic databases capable of

storing essentially all experimental data with detailed descriptions of the relevant metadata and 

Traditional methods for thermophysical data 

evaluation fail in light of the unprecedented 

growth in data availability (almost doubling 

every 10 years). Therefore, CSTL researchers 

are designing a dynamic system of 

generating evaluated data for industrial and 

scientific applications. 

164
uncertainties. The combination of these electronic databases with artificial intelligence (expertsystem) software will allow the dynamic generation of evaluated recommended data as needed. 

This concept contrasts sharply with static compilations, which must be initiated far in advance of 

need. Dynamic compilations dramatically reduce the effort and costs associated with data

programs. 

Significant advances were made in the TRC Data 

Entry Facility (to support mass-scale data entry), 

Guided Data Capture (GDC) software (to 

enhance in-house and external data entry), and 

ThermoML (an XML-based approach for 

international data collection and management). 

Full-scale cooperation between the Journal of 

Chemical and Engineering Data and NIST has 

been established in direct data communication, 

quality assurance, processing, and distribution,

based on the global use of GDC and ThermoML. 

The algorithms for the implementations of the Dynamic Data 

Evaluation concept for pure organic compounds were fully 

developed in 2003 and incorporated into the framework of the

ThermoData Engine software currently under development at 

TRC. Moreover, the targeted goal of 300,000 data points per 

year has been achieved at the TRC Data Entry Facility (for mass-scale data entry) with strict 

adherence to quality assurance. This resulted in the capture of all relevant experimental data

published between 1993 and 2003 in 5 principal journals (Journal of Chemical and Engineering

Data, Journal of Chemical Thermodynamics, International Journal of Thermophysics, Fluid Phase 

Equilibria, and Thermochimica Acta) using the TRC information systems infrastructure. To inform

customers of TRC’s progress in dynamic data generation and evaluation, the TRC Consortium 

Annual Workshop was held in FY 2003. 

TRC efforts in the development of new

XML-based technology for thermodynamic 

data communications (ThermoML) have

been recognized in the establishment of the 

IUPAC project “XML-based IUPAC Standard 

for Experimental and Critically Evaluated 

Thermodynamic Property Data Storage and 

Capture”. It is anticipated that ThermoML 

will become a prototype of this IUPAC 

standard. 

NIST Computational Chemistry Comparison and Benchmark Database (CCCBDB) 

R. D. Johnson III (838) 

As computers become faster, modeling becomes a

more valuable tool. Quantum mechanics can be used

to predict thermochemical properties. However there

are many approximations in quantum mechanical 

calculations and the uncertainty in the predictions is 

not well established. The NIST Computational 

Chemistry Comparison and Benchmark Database (CCCBDB), recently updated to 

version 9, is a website and database which

allows users to compare thermochemical

properties determined by experiment or by

quantum chemical calculations. 

CSTL researchers have 

developed a database that 

allows users to compare 

thermochemical properties from 

one species to another. 

By compiling benchmark experimental and theoretical (from 

quantum mechanics) results, the CCCBDB can illustrate the 

uncertainty in the theory by allowing comparison between

theory and experiment. The CCCBDB also provides 

developers of new computational methods a set of benchmark experimental data to test the new 

The CCCBDB serves an 

average of 27000 web pages 

per month and over 10000 

distinct hosts. As well as its 

stated purpose, the CCCBDB

is also used as a source of 

benchmark experimental data. 

http://srdata.nist.gov/cccbdb/

165
methods. The experimental and calculated data includes enthalpies of formation, entropies, heat

capacities, geometries (bond lengths, angles, rotational constants), vibrational frequencies, and

dipole moments. 

Data and calculations for several new species have been added in response to user requests.

The species include diborane (B2H6), formamide (CHONH2), and a selection of substituted

aromatics, toluene (C6H5CH3), aniline (C6H5NH2), phenol (C6H5OH), chlorobenzene (C6H5Cl), 

styrene (C6H5C2H3), and naphthalene (C10H8). New web pages are being added continuously to 

allow more comparisons and east of data retrieval. The number of calculations on all molecules 

continues to grow as does the use. 

Descriptive Terms of Particle Appearance for Search and Classification 

D. S. Bright (838) 

It is very tedious to visually search an image 

collection containing thousands of particles. 

Typically, one particle is in mind and the user 

wants to find other particles that look similar. The

images need to be indexed or described, in order 

to search them automatically. We are working on

numerical, computer-generated descriptors for 

these images, such as aspect ratio, roundness, 

and others with more obscure meaning. 

Morphology is important for particle 

classification and for determining 

particle origin and chemical 

composition. The CSTL developed 

database will make searching the 

visual collection easier and more 

accurate. 

Since the images in our trial data

set are so visually distinctive, we 

thought that common sense visual 

terms, such as “round”, “smooth”,

or “sharp edged” might make

useful descriptors that would

perform better and be more easily 

understood than the numerical

descriptors. To this end, we made a glossary of 70 terms (see sample shown), with a few

pictures, and a brief explanation with links to other terms. We also assigned these terms to a 

collection of 1600 particle images, and developed software to search the collection using the

terms. 

Flattened oblate, thicker than flake. Not bent ...

 

Typical scanning electron micrographs of particles from the collection of 1600 images. 

166
 Data Activities in FY 2003 

(Described in more detail elsewhere in this report.) 

Automotive and Aerospace (Transportation) 

Benchmark Data on Liquid Fire Suppressants for Use in Aircraft 

C. Presser and B. Johnson (836); C.T. Avedisian (Cornell University); G. Papadopoulos 

(Dantec Dynamics); J. Hewson (Sandia National Laboratory); D. Keyser (NavAir); P. 

Disimile and J. Tucker (46th Air Force Test Wing) 

Pharmaceuticals and Biomanufacturing 

A Metric of Amino Acid Exchangeability Based on Experimental Data 

A. Stoltzfus (831), L. Y. Yampolsky (CARB/UMBI) 

Biological Macromolecule Crystallization Database 

G.L. Gilliland, M. Tung, T.N. Bhat, and J.E. Ladner (831) 

Structure to Function: Using a Structural Genomics Approach 

G. L. Gilliland, J.E. Ladner (831); A. Teplyakov, G. Obmolova, O. Herzberg, J. Orban, John 

Moult (UMBI); and Andrew J. Howard (Illinois Institute of Technology). 

Chemical and Allied Products 

International Standards for Refrigerant Properties 

M.O. McLinden and E.W. Lemmon (838) 

The Industrial Fluid Property Simulation Challenge 

R. Mountain, A. Chaka, R. Johnson, D. Friend, J. Magee, L. Watts, T. Bruno, R. Chirico, A. 

Laesecke, E. Lemmon, and M. McLinden (838); J. Olson, J. Moore, and D. Frurip (Dow 

Chemical); M. Schiller (Dupont); J. Golab (BP); F. Case (Colgate-Palmolive); R. Ross (3M); 

P. Kolar (Mitsubishi Chemical) 

Transport Property Models for Refrigerants

M.L. Huber, A. Laesecke, and R. Perkins (838) 

Measurements, Modeling, and Data for Pressure-Driven Membrane Separations 

C. Muzny, H. Sun, and J. Moon (838); J. Cho (University of Colorado); M. Chapman (U.S. 

Bureau of Reclamation); and J. Ranville (Colorado School of Mines) 

Evaluated Molecular Structures and Vibrational Frequencies for C1-C2 Chlorocarbons 

D.R. Burgess, Jr. and J.A. Manion (838) 

Thermodynamic Interpretation of Low-pressure Ion-molecule Reactions 

K. K. Irikura (838) 

Evaluation of Free Radical Standard Potentials 

P. Neta and R. E. Huie (838) 

167
Energy and Environmental Technologies

Natural Gas Flow: North American Laboratory Comparison Project 

P. I. Espina (836) and W. F. Guthrie (898) 

High-Temperature Reference Correlations for the Solubility of Gases in Water 

A.H. Harvey (838); R. Fernández-Prini (CNEA, Argentina); J.L. Alvarez (CNEA) 

A Question of Accuracy: How Does Thermal-Optical Analysis for Climatically Important 

Black Carbon in Atmospheric Soot Behave Optically? 

J. M. Conny (837) 

Chemical Kinetic Database for PAH formation for Heptane Combustion 

V. Babushok and W. Tsang (838) 

OH Kinetics – Evaluation of Data and Experimental Techniques 

V.L. Orkin and M.J. Kurylo (838) 

Thermodynamics Reserch Center (TRC) Comprehensive Program on Critical Data 

Evaluation 

M. Frenkel, R.D. Chirico, Q. Dong, R.A. Stevenson, and G.R. Hardin(838); V.V. Diky (Guest 

Researcher); X. Yan, R.C. Wilhoit, and X. Hong (Contractors) 

Weakly-bound Isomers of the ClOO Free Radical 

K.K. Irikura (838) 

Expansion of NIST’s Environmental Specimen Banking Activities 

P.R. Becker, R.S. Pugh, S.S. Vander Pol, J.R. Kucklick, R.D. Day, S.J. Christopher, B.J. 

Porter, M.B. Ellisor, and S.A. Wise (839) 

Forensics and Homeland Security 

Standards for Detection of Biological Threat Agents 

K. D. Cole, L. Wang, A. Gaigalas, and D. Hancock (831) 

Health and Medical Technologies 

HIV Protease Structural Database 

T.N. Bhat, G.L. Gilliland, V. Ravichandran, and M. D. Prasanna (831); J. Vondrasek 

(NCI/NIH); A. Wlodawer (NCI/NIH) 

Human Mitochondrial Protein Database: A Resource for Human Mitochondrial Proteomics 

V. Ravichandran, P. E. Barker, G. B. Vasquez, T.N. Bhat, G.L. Gilliland (831), S. J. Zull (ATP)

Standards Requirements for Systems Biology Approaches to Health Care: Mitochondrial 

Proteomics 

Gregory B. Vásquez and Peter E. Barker (831); Steven Zullo (ATP/NIST) 

168
 Industrial and Analytical Instruments and Services 

Materials Evaluation and Database Tabulation for Fundamental Parameters and 

Algorithms Used For Quantitative Electron Probe Microanalysis 

J. T. Armstrong (837) 

Development of a New Database for the Simulation of Electron Spectra for Surface 

Analysis (SESSA) 

C. J. Powell; W. S. M. Werner (Contractor); W. Smekal (Contractor) 

Evaluation of Electron Elastic-Scattering Cross Sections 

C. J. Powell; A. Jablonski (Polish Institute of Physical Chemistry); F. Salvat (University of

Barcelona) 

SpectroML—An Extensible Markup Language for UV-Visible Spectroscopy Data 

A.D. Nguyen, A. Arslan, M.V. Smith, J.C.Travis, and G.W. Kramer (839)

Molecular Simulation of Alkylsilane Stationary Phases in Liquid Chromatography 

K.A. Lippa, L.C. Sander, and R.D. Mountain (838) 

Microelectronics 

Thermophysical Properties of Gases used in Semiconductor Processing 

J. J. Hurly, K.A. Gillis, and M.R. Moldover 

169
170
13. Technologies for Future Measurements and Standards 

... anticipating and addressing next generation measurement needs of the nation

CSTL maintains a strong basic research program in broad aspects of

measurement science that positions us with the capability to provide the

required measurements, standards, and data to support future technology 

development. New measurement techniques are important for a host of

industries including biotechnology, healthcare, environmental technologies, 

and the chemical process industry. Many of our activities, such as protein

crystallography and materials microanalysis, are at the leading edge of 

science and technology. 

 

Molecular Electronics Metrology

J. D Batteas, J.C. Garno, and S. W. Robey, L.J. Richter (837); C.A. Gonzalez (838), C.A.

Hacker (EEEL); C. D. Zangmeister and R. D. van Zee (836) 

The drive to increase electronic device 

performance, with the associated push to eversmaller device dimensions, has led industry 

observers to conclude that silicon-based

technology will reach a point of diminishing gains

in the near future. This, in turn, has generated

interest in alternative technologies based, for

instance, on single-electron devices and 

molecular components. It is hoped that the

tremendous flexibility available with organic synthetic chemistry and self-assembly techniques 

can be harnessed to produce non-linear devices analogous to silicon-based diodes and 

transistors, but comprised of single or small 

numbers of molecules. The CSTL team of

researchers in molecular electronics is 

integrating a range of techniques that will 

provide key information on electronic structure

and electron transport in candidate molecular 

electronic systems. 

Molecular Electronics (“moletronics”) is 

a field that many predict will have 

important technological impacts on the 

computational and communication 

systems of the future. In these systems, 

molecules perform the functions of 

electronic components. 

Photoemission system used to study

molecular electronic structure. 

Atomic-Force/ScannedTunneling Microscope used for 

film characterization studies. 

This work will develop measurement techniques 

and expertise necessary to understand electronic 

structure and transport found in molecular systems 

comprised of organic thin films and small 

ensembles. The overall goal is to reliably provide

experimental details which, when coupled with 

theoretical input, will help to elucidate the physical

171
mechanisms that produce device function in molecular based systems. This work is also aimed at

providing the requisite measurement protocols for such systems. 

The methodologies being employed include two-photon photoemission, which accesses 

unoccupied electronic levels and tracks electron relaxation effects, scanned probed microscopies, 

which can characterize electron transport down to the single molecule level and afford means of 

manipulating matter on the nanometer scale, as well as theoretical modeling of electronic states 

and transport properties to better elucidate the mechanisms involved in such transport function. 

Theoretical Studies of Electron Transport in Molecular Wires. 

We have continued our study of the possible mechanisms

governing electron transport in molecular wires. Particular 

attention has been focused to the electrostatics at the moleculemetal interface, which has been found to affect significantly the

transport properties of the system. In order to properly describe

the electrostatics at the interface, a novel algorithm has been

developed that efficiently computes the electrostatic potential by 

solving the Poisson equation at each cycle of the self-consistent 

field iteration based on ab initio electronic structure calculations.

A result of this calculation is shown in the figure to the right. This 

algorithm has been used to study the possible sources that lead 

to asymmetric current-voltage (I-V) curves in atomic wires as 

well as in a series of organic thiolates connected to gold

electrodes. In order to develop efficient and robust models to

gather a qualitative picture of electron conductance through

molecular bridges, a simple algorithm to compute the first-order 

expansion of the system’s Green function projected only onto 

fragments of the isolated molecule (no electrodes are included) 

has been implemented. This simple model, called the Green 

Function Condensed-to-Fragments model (GFCF) was found to 

be very useful in studying electronic transport properties of 

molecular bridges where the contact to the electrodes does not

seem to play an important role in determining the shape of I-V 

curve. This approach has been used to compute I-V characteristics of the molecule 2’amino-4,4’-

di(ethynylphenyl)-5’-nitro-1-benzenethiolate (NH2-NO2-OPE), found to exhibit “negativedifferential resistance” (NDR) behavior at 60 K. The results of the calculations are in good 

agreement with the experimental I-V curves. Calculations based on the same model predicted 

that the fluorinated derivative 4,4’-di(ethynylphenyl)-2’-fluoro-1-benzenethiolate (F-OPE) was also

a good candidate for NDR behavior. Chemists in the Nanotechnology Group at Kraft Foods 

worked on the synthesis of the molecule and very recently, scientists in NIST’s Surface and 

Microanalysis Sciences Division (837), have made I-V measurements that qualitatively agree with 

the predictions of this model. This is the first time that a theoretical prediction of the behavior of 

molecular bridges upon electrification has prompted experimental work leading to its validation. 

These results indicate the potential of theoretical tools in the rational design of electronic devices 

at the nano-scale. 

Charge Distribution and 

Electrostatic Potential 

profiles for benzene dithiolate attached to gold 

electrodes. 

SPM Directed Device Fabrication. Precisely engineered nanostructures provide a means for 

the exploration of chemical reactions under spatially well-defined and controlled environments.

Although not yet practical for high throughput applications and manufacturing, scanning probe 

lithography studies provide fundamental information on tip-surface interactions, structures, and

properties at the level of nanometers. 

Under the support of ATP, nanometer-sized patterns of copper were constructed at dimensions 

ranging from 100 nm to 400 nm via AFM-based lithography (see figure). The writing density and 

172
size of the patterns affect copper growth. Varying solution chemistry parameters, such as the 

immersion intervals and the concentration of metal salts, can control pattern sizes. Copper grows 

attern boundaries. An effective resist was found using

mercaptoundecanol, with very high selectivity observed, even at

the nanometer scale. Longer chain length alcohols (C

beyond the edges of the p

11 vs. C6) 

were more effective as resists. Preliminary results using

silanized tip coatings are promising for inhibiting copper

deposition on AFM tips during in situ experiments. Future work 

will investigate charge transport with copper overlayers on 

SAMS using conductive

probe AFM measurements 

at a metal-molecule-metal

interface. 

Pattterned Cu contacts on the 

SAM surface. Patterns range 

from 150 nm to 400 nm and are 

≈20 nm thick.

Orientational Effects on Electron Tunneling in Dodecanethiol. 

Obtaining a detailed understanding of electronic 

transport properties in molecules requires the

ability to correlate structure and transport

mechanisms. Here we are utilizing 

dodecanethiol as a “standard” test molecule to

provide reference I-V measurements. This 

molecule affords a means of probing the impact 

of molecular orientation on tunneling behavior. 

Self-assembled monolayers of dodecanethiol 

have been prepared with regions of densely 

packed molecules whose molecular axis is 

oriented ≈30o

 from the surface normal right next

to regions of lower density molecules which are 

arranged with the molecular axis parallel to the 

surface. Asymmetry in the tunneling I-V curves 

show that, in the case of the standing up phase, 

the tunneling behavior is rectified by the

asymmetry of the transport junctions, while in the 

laying down molecule, this asymmetry is almost 

completely removed (see figure to right) and

may be explained by differences in metal surface

potentials of the tip and sample surface. 

Spectroscopic Measurements. The configuration of

molecules within and the electronic structure of

electrically-active molecules are believed to be governing 

factors in molecular conductance. To investigate whether 

these molecular properties are related to the teststructure measurements, ultrafast laser spectroscopies 

have been used to study the geometry and electronic 

structure of moletronic films. The electronic structure of 

these films was studied using one- and two-photon

photoemission. 

Specifically,oligo(para-phenylene-ethynylene) thiolate 

chemisorbed on gold surfaces was studied. Within 5 eV 

of the Fermi level, four states were observed, two 

Orientation dependence on tunneling

behavior in dodecanethiol showing 

asymmetric I-V behavior in molecules 

standing up on the surface (a) vs. 

more symmetric behavior in 

molecules laying down (b). 

(- bias)

1 nm

a)

5 nm

(- bias)

b)

(- bias)

1 nm

a)

5 nm

(- bias)

b)

SH

SH

SH

SH

Energy (eV)

B

O

B*

O*

E

Evacuum

Fermi 0

1

2

3

4

5

-1

-2

-3

B*

obs Et, elec

Et, hole

O*

B*

O

B

SH

SH

SH

SH

Energy (eV)

B

O

B*

O*

E

Evacuum

Fermi 0

1

2

3

4

5

-1

-2

-3

B*

obs Et, elec

Et, hole

O*

B*

O

B

Energy-level diagram for 

OPE chemisorbed on Au. 

173
occupied (2.0 eV and 4.0 eV below the Fermi level–B/B*) and two and two unoccupied (3.2 eV 

and 5.3 eV above the Fermi level). This information is shown schematically in the energy level 

diagram. The two states closest to the Fermi level are assigned to a pi-conjugated molecular

orbital along the backbone of the molecule, and the other two are assigned to molecular orbitals 

formed by the “ortho” carbons. (See schematic representation of these orbitals in the figure.) 

From this assignment, the hole- and electron-barriers can be determined. These are,

respectively, 2.0 eV and 3.4 eV. The charge transport gap can also be estimated. It is between

3.7 eV and 5.4 eV, depending on the criteria used to determine the onset of transport. 

This work combines for the first time detailed theory and molecular scale measurements that can 

provide a basis for the complete understanding of electronic structure and transport effects in

molecular electronic systems. The foundation that these results provide not only benefits the 

development of molecular electronics applications, but also affords a groundwork for the

investigation of molecular based optoelectronic device applications. 

Future Plans: On the horizon are experiments 

aimed at probing the electrical behavior of

ensembles of molecules based on patterning to 

afford a means of assessing molecular function in

device level measurements, as well as scaling conductance properties in molecular ensembles. 

Future work will be aimed at correlating compression effects on the tunneling properties as

investigated by conducting probe atomic force microscopy. The results from these experiments 

will be compared to theoretical models and to the performance of device-prototypes fabricated

and tested by our collaborators in NIST’s Semiconductor Electronics Division in EEEL. The

photoemission work will systematically investigate the effects of chemical substitute on the 

molecular orbitals. Theoretical modeling of the transport properties and the valence structure of

these electrically-active compounds is also underway. 

This project provides detailed insight into 

the complex behavior of electronic 

transport through molecules. 

Microfluidics and the Microanalytical Laboratory 

L. Locascio, A. Henry, W. Vreeland (839); D. Ross (836); M. Gaitan (EEEL) 

The Microanalytical Laboratory project was 

established as a competence project in 1998 and 

has just completed its last year of funding. The work 

performed in this project is collaborative between the

Analytical Chemistry and Process Measurements 

Divisions in CSTL and the Semiconductor 

Electronics Division in EEEL, and involves the 

development of new techniques to improve

measurement capabilities in microsystems based on 

microfluidic technology. 

The goal of this competence project 

is to advance the knowledge and 

understanding of Lab-on-a-Chip 

devices based on microfluidics 

technology in order to promote 

higher performance and improved 

measurements in these miniaturized 

analytical systems.

To date, most of the separations performed using microfluidic systems utilize electroosmotic 

pumping for fluid flow and separate analytes on the basis of their electrophoretic mobilities. While 

this mode of separation is a useful one for many bioseparations, scenarios arise in which it is 

necessary to separate neutral analytes. For these separations, it is necessary to incorporate a 

stationary phase in the microchannel; the analytes can be flowed through the microchannel using

electroosmotic pumping but the basis of the separation is the interaction of the analytes with the

stationary phase. This mode of separation science is known as capillary electrochromatography 

(CEC) and is the hybridization of capillary electrophoresis and high-performance liquid

chromatography. With CEC, different species interact with the stationary phase for different

periods of time; thus, a separation of the neutral species occurs. This year, we have focused

174
some of our effort on the development of microchip-based CEC (μCEC) devices fabricated using 

polymer laser ablation. 

Transmission infrared spectrum at

Brewster’s angle of poly(styrene) 

spin-coated in Si. 

4000 3500 3000 2000 1500 1000

δ(CH)

ip

1153 cm-1

(CH2)

wag

1318 cm-1

ν(ring)

1492 cm-1

ν(ring)

1584 cm-1

δ(C-H)

oop

1742 cm-1

δ(C-H)

oop

1799 cm-1

δ(C-H)

oop

1872 cm-1

ν(CH2)

sym

2848 cm-1

δ(CH)

oop, ip

1067 cm-1

δ(CH)

ip

1180 cm-1

δ(CH)

ip

1029 cm-1

(CH2)

wag

1371 cm-1

δ(CH2)

1454 cm-1

ν(ring)

1601 cm-1

ν(O-H)

3436 cm-1

ν(CH2)

asym

2927 cm-1

δ(C-H)

oop

1943 cm-1

ν(C-H)

arom

3104 cm-1

3083 cm-1

3061 cm-1

3026 cm-1

2998 cm-1

{

Pristine PS

0.005 AU

Absorbance

Wavenumber (cm-1)

4000 3500 3000 2000 1800 1600 1400 1200 1000

PS ablated in H2SO4

0.005 AU

ν(O-H)

3272 cm-1

ν(C=O)

1723 cm-1

Absorbance

Wavenumbers

Transmission infrared spectrum at

Brewster’s angle of poly(styrene) 

spin-coated in Si and ablated under 

H2SO4 vapor. 

The laser ablation of polymeric materials results in a microchannel surface that is significantly 

rougher than embossed microchannels. Additionally, the chemical species present on ablated 

surfaces are much different than those present on a pristine or embossed polymer substrate, and 

can be modified by changing the atmosphere under which the channel is ablated. The figures 

show the transmission infrared spectra at Brewster’s angle of poly(styrene) (PS) spin-coated on 

Si, and PS ablated in an atmosphere of H2SO4 vapor. The two spectra are quite different with the

H2SO4-ablated PS spectrum showing bands indicative of carboxylic acid species suggesting that

this channel would have a higher density of negative charges at pH values > ~4. Because 

electroosmotic flow (EOF) rates increase with increasing surface charge density, we expect a 

higher EOF in carboxylate-terminated PS surfaces than in their pristine counterparts. 

2 4 6 8 10 12

-2

-1

0

1

2

3

4

5

6

EOF (c

m2/ V sec)

pH

 Pristine PS

 PS ablated in NH3

 PS ablated in air

 PS ablated in O2

 PS ablated in N2

 PS ablated in He

 PS ablated in H2SO4

The figure shows the dependence of EOF on

pH for microchannels ablated under a variety of

atmospheres. 

The microchannel ablated in H2SO4 supports 

the highest EOF, indicating higher density of

charged moieties on this microchannel surface.

While the presence of charged species on 

microchannel surfaces is essential to drive 

EOF, it is also important for the covalent

addition of stationary phases to the

microchannel surfaces for μCEC separations.

We have modified H2SO4 ablated

microchannels to link various stationary phases 

suitable for μCEC, and experiments are now 

underway to use those stationary phases in the 

first PS-based device. 

In this project, we strive to produce critical 

fundamental data on flow control and

manipulation that underpins device 

performance in these miniaturized flow systems. This work is comprehensive involving microflow 

Measurement of EOF using current

monitoring method in PS microchannels 

fabricated by laser ablation in various 

atmospheres. 

175
measurement, temperature measurement, flow modeling, chemical surface modification, and

materials characterization. We are continuing collaborative work with CFD Research Corporation 

(initiated last year) on the development of microflow models for polymer microchannels that will 

be ultimately applied to predict and optimize results of separations performed in polymer-based

systems. In the past 5 years of this competence building project, we have produced

approximately 50 publications with 8 patents/invention disclosures. In the next year, we plan to

become more involved in the newly formed standards activities groups to support 

microtechnologies. We also will initiate a new effort with the National Institute of Justice for the

development of microsystems based for DNA forensic analysis. Our research efforts in the

development of novel analytical methods for microchemical separation and detection will continue

to be a major part of our program. 

We are also continuing the development of temperature gradient focusing methods for 

microanalytical separation and pre-concentration. This year, the method was extended to the 

separation and concentration of neutral and chiral analytes. 

Temperature Gradient Focusing for Biological Assays in Microchannel Systems 

K.M. Balss, D.J. Ross, M.J. Tarlov (836); H. Begley (BFRL), K.G. Olsen (Loyola College) 

See Health and Medical Technologies for details.

Our work continues to involve several active 

collaborations both internally (EEEL, PL, CSTL, and 

MSEL) and externally (Virginia Tech, Cornell,

Stanford, Clemson, CFD Research Corporation, 

University of North Carolina, University of Maryland.

For example:

In FY 03, we have developed essential

collaborations for the propagation of 

fundamental data and measurement 

methods. We have also developed 

several key collaborations for the 

development of systems that have

application to NIST strategic focus 

areas including Homeland Defense, 

Nanotechnology and Health Care. Cell-based Sensors for Screening Toxins 

L. Locascio, J. Travis (839); 

Suggs, B.J. Love, N.G Love (Virginia Polytechnic 

and State University) 

See Forensics and Homeland Security for details.

In FY 03, we developed methods to enhance mixing in microfluidic formats, and also introduced

methods for mixing based on selective release of reagents from liposomes see details below. 

Microfluidics: Rapid Mixing in Microfluidic Channels 

W. Vreeland and L.E. Locascio (839) 

The liposome is a spherical structure composed of 

a phospholipid bilayer membrane that encapsulates 

a volume of intravesicular aqueous solution. 

Liposomes are bathed in an external aqueous 

solution and are generally ~100 nm to ~1 μm in 

diameter. The ability to encapsulate a species of 

interest inside liposomes renders that species inert 

to chemicals residing outside of the membrane. 

We have developed a bio-inspired liposome system 

that allows for the controlled introduction of polar species into a microfluidic system through the 

modulation of temperature using thermally triggerable liposomes. Thermally triggered liposomes 

take advantage of the dramatically increased bilayer permeability near the lipid chain melting 

transition temperature (Tm). Thus at a controlled temperature, a thermally triggerable liposome

will release its contents into the extravesicular microfluidic space, allowing for precise delivery of 

agents to specific regions in the microfluidic environment. 

CSTL researchers have employed 

lipid vesicles to mix and deliver 

reagents in microfluidics systems 

with exquisite rapidity and control, 

and have demonstrated reagent 

mixing approximately five times 

faster than standard passive mixing.

176
This phenomenon is shown in the figure, where a 

solution of thermally triggerable liposomes 

(composition 97 % dipalmitoylphosphatidylcholine 

+ 3% cholesterol) encapsulating self-quenched 

fluorescent sulforhodamine B flow through a 

microchannel with a lateral temperature gradient. 

False-color micrographs of the fluorescence

intensity of a solution of 97 mol% DPPC, 3 mol%

cholesterol liposomes encapsulating selfquenched 100 mM sulforhodamine B in 0.5 M Tris 

buffer flowing through a polycarbonate 

microfluidic channel under different applied 

temperature gradients of 20°C-40°C to 40°C-80°C 

over 2 mm distance at a flow rate of 5 μL / hour. 

The left end of the channel is thermostated at 20 °C and the right side is thermostated at 

temperatures ranging from 40 °C to 80 °C in 5 °C increments; please note that due to the low 

thermal conductivity of polycarbonate, the magnitude of temperature gradient in the channel is 

slightly less than the nominal temperatures at the termini. As the liposomes flow from the lower 

temperature region of the channel to the higher temperature region, they pass through the Tm that

is specific for this formulation of liposomes (36  ̊C in this demonstration). At the Tm, the

liposomes release the fluorescent dye into the extravesicular space causing an increase in

fluorescence at that point and downstream in the channel in all panels except the first, where the

temperature gradient does not pass through the Tm of this liposome formulation. Thus, the

fluorescent dye, which was initially segregated from the extravesicular space, is effectively

“mixed” into the microfluidic environment. Because the liposomes are dispersed throughout the 

channel, mixing upon release from the liposomes is inherently quite fast. Measurement of the 

profiles in the figure show the dye goes from “unmixed” to “mixed” state in approximately 200 μm 

under the experimental conditions shown here, whereas standard diffusive mixing would require

almost 9 cm. Since this “mixing” technique is controlled by the Tm of the liposome formulation,

rather than the fluid mechanical properties of the channel, this technique should be less sensitive 

to different operational flow rates than traditional microfabricated mixers. Additionally, because 

the liposome formulation dictates the Tm, mixed populations of liposomes can be used to control a

series of sequential reactions where the temperature in the channel determines reaction timing 

and sequence. 

All other techniques for enhanced mixing efficiency in 

microfluidic systems operate optimally in certain fluid

mechanical regimes; our liposome-based system,

however, depends on the heat transfer and 

thermodynamic parameters of microfluidic device and

thus is limited by a different set of criteria. This is 

crucial to our effort to design and demonstrate 

microfluidic systems that are both robust and efficient. 

Future work on this project will focus on using

liposomes to act as packages to study individual biomolecules, such as single-molecule proteins. 

Since the permeability of the lipid membrane can be easily modulated through temperature

control, various species can be presented to the biomolecule in a controlled fashion to study the

effects of that agent on individual, single biomolecules. 

This work is the first demonstration 

of a mixing strategy in microfluidic 

systems that is not dependent on 

the operational flow-rate. This 

system is ideally suited for 

biological analyses as the liposome 

itself mimics the natural 

environment of the biological cell.

177
Nanobiotechnology: Nanofluidics and Nanoscale Chemical Reactions 

L.E. Locascio, W. Vreeland, K. Brazhnik (839); R. Kishore, S. Kulin, K. Helmerson (PL) 

This project focuses on the development of nanoscale

structures to facilitate the performance of ultra-small

volume chemical reactions and separations. The 

nanoscale structures that we are designing are 

composed most often of phospholipid molecules and

are self-assembled spherical or tubular structures with

diameters ranging from tens to hundreds of nanometers. 

We have demonstrated the use of these nanometer 

structures for controlled chemical reactions using

picoliters of reagents. 

The work described here is 

associated with the Single 

Molecule Manipulation and 

Measurement competence 

program whose purpose is to 

study the behavior of 

biomolecules one molecule at a 

time to elucidate the differences 

that make them uniquely 

beneficial or detrimental.

Due to their amphiphilic nature, when phospholipid molecules are dispersed in water they selfassemble into bilayer membranes to form structures called liposomes that are often spherical and 

encapsulate an aqueous internal volume. 

Liposomes range in size from 50 nm to 10’s 

of micrometers encapsulating volumes that 

are measured in attoliters to picoliters. 

Water-soluble molecules, including DNA,

RNA, proteins and small ionic molecules,

can be readily incorporated into the

liposomes upon formation. In this work, we use liposomes to sequester very small amounts of

reagents in discrete packages to control their reaction. The liposomes are trapped and 

manipulated in a microenvironment using optical tweezers. Liposomes containing two different 

reagents that are brought into contact using the optical tweezers do not bind or fuse, therefore

their contents cannot react. However, if the liposome membrane is exposed to a short UV laser

pulse while in contact with another liposome, the membrane destabilizes and the two liposomes 

fuse together to form a single liposome structure as shown in the figure above. During fusion, the 

contents of the two liposomes react immediately. The time scale of liposome fusion events varies

in length from milliseconds to seconds 

and is most likely dependent on the

lamellarity of the liposome membrane. 

a.Two liposomes brought into contact

using optical tweezers. 

b. Laser pulse at the interface initiates 

fusion of the two membranes. 

c. C, D. Fusion is completed in the next

2 successive video frames. 

Phospholipids can be forced to form nonspherical structures under certain

conditions. We recently reported the 

formation of phospholipid tubes in a 

microfluidic device under conditions of 

high shear. Above the phase transition

temperature of the phospholipid, lipid 

hydration in the microchannels resulted in 

self-assembly of lipids into tubes with

diameters of several hundred nanometers to a few micrometers and lengths of up to several 

Phospholipid tubes formed in a microchannel 

under conditions of high shear under flow. Tubes 

are oriented in a direction parallel to the long axis

of the microchannel. 

178
centimeters. In general, tubes aligned parallel to the lateral dimension of the microchannel.

Elasticity of tubes was assessed via secondary manipulation with optical tweezers and with an 

optical scalpel. It was determined that lipid tubes formed and stored in a microfluidic setup 

became mechanically rigid after a few days of storage at room temperature or after a month of

storage at 4 o

C. 

The work described here is critical to our efforts in the development of new tools for observing

and characterizing single molecule behavior. The characterization of single biomolecules, rather

than the study of ensembles of biomolecules, is an important topic in the field of biology since it

has been elucidated that the presence and behavior of the biological outlier or the mutant version

of the biomolecule can facilitate amplification of that species resulting in catastrophic 

consequences as highlighted in recent reports on prions. Although our efforts in this area are

new, we have made considerable progress toward packaging single molecules, performing

controlled reactions with a few molecules, and designing nanochannels through which we will 

attempt to manipulate and characterize single

DNA molecules. Future work will involve 

further characterization of the lipid nanotubes 

to determine stability and lifetime. In the next

few years of the competence program, we

plan to pursue the incorporation of lipid 

nanotubes into higher-level microsystems and

apply these toward the study of single DNA 

and RNA molecules. 

Nanowire devices will provide multiple options as 

building blocks for sensors, miniaturized 

measurement platforms, and as advanced scatter

probe structures, enabling true nanometer 

spectroscopy. This methodology will provide an 

inexpensive and straightforward approach to the 

synthesis of sensing devices with multiple 

functionalities that can be integrated to form

state-of-the-art measurement systems. 

Improved Vacuum Transfer Standards – Spinning Rotor Gauge Stability Studies 

R. F. Chang (836) 

NIST/CSTL seeks to disseminate its realization of the 

Pascal at high vacuum levels directly to our 

customers and into readily available commercial 

gauges. However, commercial vacuum transfer 

gauges are not sufficiently stable to effectively 

disseminate NIST’s measurement uncertainties to 

our customers. Understanding the sources of these 

instabilities is important to gauge users, as well as to

primary standard laboratories that rely upon high

stability for maintaining their Quality Systems and for 

satisfactory performance as transfer standards 

The spinning rotor gauge has 

become the transfer standard of 

choice for vacuum calibrations 

from 10-4 to 1 Pa due to good 

calibration stability (changes < 

0.5%/year). While internal NIST 

SRGs exhibit better long-term 

stability, some calibration 

customer gauges have shown 

inexplicably larger shifts.

179
Earlier we had observed that de-mounting

and re-mounting of the SRG suspension head

may cause an average shift of ~ 0.1% in

accommodation coefficient (which is directly

tied to calibration stability), σ, and in some

case as large as 0.4%. However, this did not

fully explain observations the 1% and larger

shifts in customer calibrations. In FY03, we 

investigated the effects of various handling

procedures that affect surface properties of

the rotor, such as their rinsing in cleaning

solution, weighing and measuring, mounting

and inspecting under a microscope, and even

rolling and shaking of the rotor in a thimble to 

simulating transit between laboratories. 

Surprisingly, these procedures shifted σ only ~0.25% on average, although customer shifts as

large as 0.8% were observed. These and other factors suggest that the large observed shifts in σ

are partly a manifestation of a rotational-axis-dependence of σ. Inspection of customer rotors

revealed many with non-uniform blemishes (e.g., scratches, corrosion), and some with polar 

orientations. A series of experiments using deliberately longitudinally-scratched rotors showed an

average shift in σ of 0.5%. Shift values ranged from -1% to 1%, as shown in the figure, when the

suspension head was de-mounted and re-mounted. Each new suspension has a different

rotational axis for the rotor. The extent of the resulting variability is close to that observed from 

calibration customers. Three check standards, undisturbed between calibrations, were calibrated 

along with the scratched rotors. The check standards reproduced σ each time as expected. The

data of normal rotors, which have some blemishes but without deep longitudinal scratches, are

also shown for comparison. 

-1.5%

-1.0%

-0.5%

0.0%

0.5%

1.0%

1.5%

Gauge No.

Change in 

Accommodation Coefficient

Check Standards

Scratched Rotors

Normal Rotors

1 2 3 4 5 6 7 8

Results suggest a few areas of 

exploration for improved stability of 

SRGs. For instance, we need to explore 

the manufacturing processes of the 

rotor such that a preferred rotational

axis may be established, and the 

surface is mechanically and chemically

hardened to resist corrosion and 

scratches. Choice of soft materials for 

the thimble or treatment of its inner 

surface may also aid in reducing the 

probability of scratches on the rotor 

due to sudden loss of suspension. 

We have completed a first-ever, comprehensive 

investigation into the causes of calibration instability 

of SRGs, and the findings have refuted many longheld beliefs. Our results can significantly affect the 

manufacturing processes as well as routine

operations and maintenance of SRGs. There are 

implications for new material selections as well as

input into the next generation of SRGs, underway at

both NIST and the Forschungszentrum-Jülich in

Germany. 

Johnson Noise Thermometry 

W. L. Tew (836); S. W. Nam and S. Benz (EEEL) 

JNT is the only established thermodynamic method in

contact thermometry which remains practical for 

applications above 800 K. Johnson noise temperature 

probes can be designed to sample the thermal noise from 

noble-metal alloy resistance elements up to at least 1800

K; such elements are relatively insensitive to changes 

caused by thermal strains or diffusion of impurities. The

NIST JNT technique represents an important innovation by

applying Josephson-based voltage standards to create an 

intrinsically calculable noise source, the Quantized Voltage

Johnson Noise Thermometry 

(JNT) is a primary technique 

that is well suited to certain 

demanding application 

environments and as a hightemperature contact method 

for thermodynamic 

determinations relative to the 

International Temperature 

Scale of 1990. 

180
Noise Source (QVNS). This approach conveys key operational advantages over conventional 

JNT methods already in use at other National Metrology Institutes (NMIs), and allows a fixedpoint-independent method of measuring SI temperatures. 

For NIST temperature metrology, the new JNT system is

competitive with radiation-based methods for T<1235 K. For

industrial applications, the NIST method could be tailored to the 

use of solid-state noise references for those application 

environments where thermometric stability is critical and 

serviceability is limited, such as power generation plants and 

chemical processing applications. This will benefit all users of 

temperature instrumentation who are engaged in accuracy-critical applications limited by artifact 

thermometer performance. 

The new NIST technical 

approach is being 

developed to help move

JNT into wider use in both 

the industrial and 

metrology domains. 

New circuit designs of the Josephson QVNS have

dramatically reduced the common mode voltage error 

measured by the Johnson noise measurement electronics.

This has enabled a significant improvement in the 

demonstrated accuracy of new NIST methodologies. The

new accuracy benchmarks are relative to ITS-90

assignments for the verification test fixed points: 1) the 

gallium triple point (GaTP, 302.916 K) ; and 2) the water

triple point (WTP, 273.16 K). These fixed points establish

ITS-90 temperatures that are compared to the JNT-derived

temperatures. The latest data demonstrate an uncertainty 

of 250 μK/K in the

absolute mode and

100 μK/K in the relative 

mode for the T(GaTP)/T(WTP) ratio. These results are within

the current combined estimated uncertainties for those

measurements. For the relative mode data, the agreement

with the ITS-90 value is within the JNT statistical precision

alone as estimated by the available data. 

Magnified image of a section of a 

Nb-PdAu-Nb Josephson array.

Construction has begun on a low-noise comparison furnace

for use between 273 K and 934 K that will enable

comparisons of JNT-based temperatures with ITS-90 over 

that range. The comparison furnace will be installed in the

Division 836 electrically shielded room facility. A second set of 

digital correlator electronics has been built to allow two

systems to be operated, one in Boulder and the other in Gaithersburg. 

A pseudo-noise trace as 

produced by the QVNS with 

a 1.6 kHz repetition rate 

The achievement of an uncertainty of 0.001 % in the relative mode will be competitive for 

temperature metrology applications above ∼700 K. New thermodynamic data in the overlapping

ranges of contact and radiation-based thermometry between 700 K and 1235 K would be a 

significant input for the next international temperature scale. In certain industrial areas, JNT

systems based on solid-state noise references could eventually reduce the cost-of-operation of 

any process that is limited by the calibration stability of artifact-based thermometers. Here 0.1 %

to 0.01 % uncertainties are sufficient, depending on the specific application. 

CSTL researchers plan to use the JNT prototype system with a QVNS, to determine the 

differences between JNT-determined temperatures and temperatures on the ITS-90 over the

range of the comparison furnace (273 K and 933 K). EEEL researchers will continue to refine the 

QVNS and development of a custom programmable arbitrary bit stream generator, a key 

component to improving noise and other waveform synthesis capabilities.

181
Atomic Standard of Pressure 

M. Moldover and J. Schmidt (836); K. Szalewicz (U. Delaware); Y. Wang (EEEL)

CSTL researchers are developing a novel primary standard for pressure in the range 0.3 MPa to 

5 MPa. The new standard will determine the pressure p(ε,T) by measuring and calculating the 

dielectric constant ε(p,T) of helium with extraordinary accuracy. The uncertainties from electrical 

and temperature measurements will be smaller than the uncertainty of existing pressure 

standards (piston gages).

This standard is being developed to: (1) replace artifact-based

pressure standards with a standard based on calculable

properties of a pure substance: ε(p,T) of helium gas and; (2) 

reduce the uncertainty of pressure standards in the range 300 

kPa to 5 MPa. Below 300 kPa, the primary pressure standard at

NIST is a mercury manometer. Above 300 kPa, the pressure

standards are commercially manufactured piston-cylinder sets. 

These sets are complicated artifacts. In operation, the cylinder 

and piston deform significantly and the piston rotates 

continuously to insure gas lubrication. Because of these 

complications, piston-cylinder sets are calibrated against the

primary-standard mercury manometer below 300 kPa and their

performance is extrapolated to higher pressures using numerical

models of the coupled gas flow and elastic distortions. Pistoncylinder sets exhibit a poorly-understood specie and gas flow

dependencies. Thus, the extrapolation is not fully trusted and it

cannot be checked with existing technologies. When ε(p,T) of

helium becomes the pressure standard, it will be possible to test 

models of piston-cylinder sets and to reduce their uncertainty. 

Section of cross capacitor 

and components. 

Dielectric constant measurements are being improved

by drawing on NIST’s expertise in electrical metrology.

Using that expertise, we developed a novel, doughnutshaped, four-electrode, cross capacitor. In comparison

with conventional capacitors, cross capacitors are more

stable and less subject to surface contamination

(oxides, adsorbed water, or films of oil). Toroidal 

capacitors have an additional advantage; there are no 

“end effects” to complicate measurements. During 

FY02/03, a toroidal cross capacitor and a rod-shaped 

cross capacitor were used to measure the dielectric 

constant of helium in the range 0 to 7 MPa. The

measured values of ε(p,T) from the cross capacitors of

differing designs were consistent with the theoretical 

values and with each other within an uncertainty of 

2×10-7 which corresponds to a relative pressure 

uncertainty of 5×10-5 which is approximately an order of

magnitude larger than current pressure

standard uncertainties. Quasi-Spherical Microwave Cavity. The cavity

is assembled from hollow, copper, quasihemispheres. Each quasi-hemisphere has the 

shape of two quadrants of a sphere (radius = 

5 cm) with 0.01 cm wide cylindrical extensions 

(exaggerated in the sketch). The extensions 

split degenerate microwave frequencies. 

The theory for ε(p,T) is being advanced at the 

University of Delaware, and a concept for a

more stable cross capacitor is shown in the 

figure. This capacitor is made from a single,

182
long, sapphire crystal. The crystal is 

edge-grown, with a hollow, star-shaped 

cross section. It has four surfaces that 

are coated with thin electrodes. We 

expect that the single-crystal sapphire 

capacitor will be more stable than 

conventional cross capacitors assembled 

from many metal and insulating parts. To

advance capacitance bridge technology 

an order of magnitude, NIST has let a 

SBIR contract to a bridge manufacturer. 

In parallel, NIST’s EEEL is developing the technology to test such a bridge when it is delivered. It

is not certain that capacitance bridges can achieve the desired performance at a reasonable

price. Thus, CSTL is exploring the alternative of making very accurate measurements of ε(p,T) of 

helium at microwave frequencies. Our approach uses quasi-spherical microwave cavities. This 

concept grew out of CSTL’s long experience in developing and using of spherical cavities for 

acoustic thermometry. 

To reduce the uncertainty a factor of ten, we are 

simultaneously attacking the problem on four 

fronts: 

(1) improve the theory for ε(p,T) and the theory

for the virial coefficients of helium, 

(2) manufacture cross capacitors with greater

stability and larger capacitances, 

(3) obtain a more accurate capacitance bridge 

for measuring ε(p,T) at audio frequencies, 

(4) measure ε(p,T) with high resolution at

microwave frequencies. 

If successful, this program will revolutionize pressure 

standards and greatly improve capacitance bridges. 

Already, CSTL has used cross capacitors to measure

ε(p,T) of the primary constituents of natural gas 

including methane, ethane, propane, nitrogen, carbon

dioxide, and argon. The measurements span the 0oC 

to 50oC temperature range and extend to 7 MPa. In

this range, they are more accurate than any previous 

measurements and provide reference data for use in

metering natural gas. 

Sketch of single crystal sapphire cross 

capacitor. Thin-film electrodes are colored 

blue and red. The prototype is 25 cm long 

and 2 cm across the largest “diameter”. 

Improving Limits of Detection in Microanalysis with High Speed Energy Dispersive X-Ray 

Spectrometry (silicon drift detector EDS) 

D. E. Newbury (837) 

A critical issue in many applications of electron beam x-ray microanalysis is achieving a low value 

of the concentration limit of detection, expressed

as CMMF (minimum mass fraction). The formula for 

CMMF incorporates with equal weight two critical 

spectrometry terms, the peak counting rate, P, and

the spectral peak-to-background ratio, P/B. The 

P/B is a direct consequence of the physics of

generation of characteristic and continuum 

(bremsstrahlung) x-rays and the resolution of the 

spectrometer. For a given target composition and 

beam energy, the measured P/B is effectively 

limited by the choice of the spectrometer. Thus, the high-resolution wavelength dispersive

spectrometer (WDS) provides a better (lower) value of CMMF than the conventional silicon energy 

dispersive spectrometer (Si-EDS) for equivalent peak counting rates on a particular peak. 

However, when many trace constituents must be evaluated at each analyzed point, the capability

of an energy dispersive spectrometer to simultaneously evaluate the entire excited energy range

The performance of a new silicon drift 

detector (SDD) EDS capable of much 

higher count rates has been evaluated

in terms of the CMMF attainable. Trace 

element constituents (concentration < 

0.01 mass fraction) have been 

detected in less than 10 seconds of 

spectrum accumulation time. 

183
of x-rays provides a substantial time advantage over the WDS, which must be scanned 

sequentially over this range with several diffractor changes. In this case, to compensate for the 

inherently poorer P/B of the EDS, it becomes advantageous to increase the peak counting rate, 

P. Conventional Si-EDS systems are limited to total spectrum output count rates of

approximately 25 kHz. 

The silicon drift detector (SDD) energy dispersive 

x-ray spectrometer, developed by Radiant

Detectors LLC of Northridge, CA under NIST SBIR grants, is capable of a much higher counting

rate than the conventional Si-EDS. In an SDD, the collection of the charge deposited during

photoelectric capture of an x-ray photon is much faster due to (1) a shaped internal electrical field 

created by a lithographically applied electrode pattern on the back surface; (2) the reduced

thickness dimension of the SDD (300 m compared to 3 mm for Si-EDS); and (3) the higher 

temperature of operation (250 K vs 

77 K for Si-EDS) for faster charge

carrier drift velocities. Total 

spectrum counting rates in excess of

500 kHz have been achieved, 

limited by the digitization of the data

stream not the SDD detector 

performance. 

The SDD can achieve CMMF values

of below 10-3 mass fraction in short

spectrum accumulation times, e.g., 

10 s. The figure shows an example

of a 10 s spectrum of copper 

recorded at a total spectrum output 

count rate of 164 kHz (input count

rate ~300 kHz). 

nsity Inte

Silicon Drift Detector

Time constant = 750 ns

(236 eV FWHM at MnKα)

Output count rate: 164 kHz

Cu Beam energy E0 = 20 keV

Time = 10 s

FeKα

C =0.0040 mass fraction

Photon energy (keV)

Two examples of trace

measurements are shown: the 

detection of iron at a level of 0.0040

mass fraction, and a CMMF value established for manganese at 0.00092 mass fraction. For a 

focused beam of 20 keV electrons incident on copper, the total sampled mass corresponds to 16 

picograms of copper. Within this matrix mass, the trace iron constituent corresponds to the

detection of 63 femtograms, while the concentration limit of detection for manganese corresponds 

to 14 femtograms. 

MnKα

CMMF =0.00092 mass fraction

FeKα

CuKα CuKβ CuL

Copper target excited with 20 keV electrons and 

measured with a Radiant Detectors LLC SDD x-ray

detector. The spectral regions for manganese (Mn Kshell) and iron (Fe K-shell) are indicated. 

An operational SDD detector has been

successfully demonstrated in the 

spectrum acquisition mode to significantly 

lower the limit of detection in point beam 

analysis. However, the real promise of

the SDD lies in high-speed x-ray 

mapping. X-ray mapping with the

conventional EDS, while widely used, is 

subject to great time penalties, running to

hours, if the analyst wishes to map minor 

constituents (0.01 to 0.1 mass fraction) or

even longer for trace constituents (< 0.01

mass fraction). The SDD has the 

capability, in principle, to achieve efficient

and effective x-ray spectrum imaging, in 

The conventional monolithic semiconductor EDS 

device was invented in the U.S. in the 1960s, and

U.S. manufacturers dominated the market until the 

1980s, when foreign competitors gained a 

significant market share. The SDD is now

emerging as a “killer application” which threatens 

to sweep away virtually the entire market for 

conventional monolithic semiconductor EDS 

devices since the SDD doesn’t require constant

liquid nitrogen service and the SDD has been

demonstrated to be capable of both higher 

resolution at long time constant (125 eV for SDD 

instead of 129 eV at MnKαfor Si-EDS) as well as 

much higher count rates at short time constant 

(500 kHz to 1 MHz for SDD compared to 25 kHz for

monolithic Si-EDS 

184
which an entire x-ray spectrum is saved at each picture element (pixel) of a map with a short 

dwell time, e.g., 100 ms. X-ray spectrum imaging can greatly improve the utility of mapping by 

recording comprehensive “flash” maps requiring 10 minutes or less to accumulate but still 

capable of mapping minor and trace level constituents. The current limitation to this process is the

high rate digitization that is required. Existing digital pulse processing systems become unstable 

at approximately 200 - 400 kHz when the full spectrum must be sampled with a short integration 

time (e.g. 10 ms). More research is planned in this area to extend the maximum digitization rate

to higher values. 

The SDD can have other impacts throughout x-ray spectrometry. A separate SBIR grant to 

Radiant Detectors LLC has demonstrated that the SDD can serve as the detector for x-rays in a

diffractometer, providing distinct advantages over the conventional gas detector by eliminating the 

need for a monochromator, since the energy resolution of the EDS is sufficient to distinguish the 

CuKα and CuKβ x-rays from a copper target used to scatter off the crystalline target. A closely 

related application is the replacement with an SDD of the gas flow proportional counter in a 

wavelength-dispersive x-ray spectrometer (WDS) used on the electron microprobe. By using the 

SDD as a high-speed counter, the WDS would become compatible with medium vacuum 

operation (10-6 Pa). The energy resolution of the SDD will also permit easy separation of different 

photon energies diffracted to the same Bragg angle, n λ = 2d sin θ where n is the integer order, 

λis the x-ray wavelength, d is the crystal plane spacing, and θ is the Bragg angle. When the 

product n λ is the same for two different photon energies, both are recorded at the same

diffraction angle. The SDD would show these components as two well-separated peaks. 

New Smart Gel Mechanism Elucidation from Immersive Visualization of Molecular 

Simulation 

C.A. Gonzalez (838); S. Satterfield (ITL); Y. Simon-Manso (Universidad de Chile); Y. Aray 

(Instituto Venezolano de Investigaciones Cientificas); Manuel Marquez-Sanchez (KRAFT 

Foods, Inc.) 

“Shake gels” are a class of materials resulting from 

an aqueous solution of a clay (Laponite) and an

associative polymer such as polyethylene oxide 

(PEO). These watery mixtures of clays and 

polymers, firm up into gels when shaken, and then 

relax again to the liquid phase after some time has 

passed, however the process is complex and as yet unknown.

The main objective of this work is to elucidate the mechanisms 

governing the gelling processes in “shake gels”, and to provide 

reliable predictive tools to guide experimentalists in the rational 

design of novel “shake gel” systems tailored to different 

industrial applications. In particular, we are interested in

understanding the nature of the interactions between the 

different components of the “shake gel” (water, laponite and

PEO) and the effects of the shaking in the gelling process. To

achieve this goal the information obtained from theoretical 

calculations were analyzed with a 3-D immersive visualization

engine available at ITL, which allows scientists to immerse

themselves among giant-sized molecules whose behavior can be seen and understood in

minutes instead of the weeks required using traditional techniques. The work described here will 

develop the necessary theoretical infrastructure to aid in the elucidation of the complicated

mechanisms involved in the formation of “shake gels”. 

Scientists in industry are currently

working on the application of “shake 

gels” in the manufacture of novel 

biological fluids, food thickening 

agents, drug delivery materials, 

cosmetics, and sensors.

185
Making use of a novel algorithm developed at the 

Instituto Venezolano de Investigaciones Cientificas to

determine the topology of the electrostatic potential of 

molecules interacting with surfaces, we were able to 

determine the mechanisms by which PEO interacts 

with the clay surface and water in order to produce

the gel. According to this mechanism, it is the

polymer's oxygen atoms, instead of its hydrogen 

atoms as previously thought, that attach to the clay.

In addition, electrical charges affect the binding 

process, resulting in water binding to clay surfaces in

a perpendicular arrangement, which is believed to help create the firmness of the gel. Our results 

also indicate that the polymer size is important. Thus, our calculations predict that larger or 

smaller polymers cannot undergo the conformational changes necessary to optimize the binding 

to the clay. These predictions are in very good agreement with experimental findings that “shake 

gels” have only been observed with solutions of PEO. 

This research provides important

mechanistic details about the 

electrostatic interactions between

water, PEO and Laponite not previously

known. For the first time, detailed 

quantum chemical calculations and 

atomistic simulations are integrated in 

order to gain a more complete picture of

the physical and chemical properties of 

these gels. 

The next step of this program will involve a

systematic study of the role of the “shaking” and

temperature in the gelling process. Once a better 

picture of the role of these two factors is obtained, 

the models will be used to guide experiments in the

synthesis of other “shake gels” with different 

chemical and physical properties that might be useful 

for industrial applications (for example: optical 

properties, viscosity, etc.). 

Universal Validation Standard for UV/visible Spectrometry 

D.L. Duewer and J.C. Travis 

Details provided in the Exploratory Research section.

Growth of Oriented ZnO Nanowires on Sapphire and Gallium Nitride 

Babak Nikoobakht and Stephan J. Stranick (837)

Details provided in the Exploratory Research section. 

Progress Towards Development of an Improved Method of Phase Identification by 

Selected Area Electron Diffraction

S. Turner, D.S. Bright (837); and V.L. Karen, (MSEL) 

Details provided in the Exploratory Research section. 

186
C. Exploratory Research

Since 1999, the CSTL laboratory director has invited all technical staff to submit Exploratory 

Research Proposals. The insights gained from these proposals have become an integral part of

our strategic planning, and a mechanism to engage all staff scientists in this important process. 

Scientists are encouraged to begin to discuss ideas among themselves, and where appropriate,

combine the ideas into a single proposal. Over the past five years 67 exploratory research

projects have been funded, and CSTL has invested close to $5M. More importantly, we have

been able to provide seed money for critical areas of research such as new approaches to

modeling, measurement methods, or data development and dissemination, as well as novel

mechanisms to provide reliable and timely measurement traceability to our customers. The work

described in the following pages is a result of the research from FY 2003 funded projects. 

Absorption Coefficient Measurements of Aerosol Particle Agglomerates 

C. Presser (836); J. Conny (837); A. Nazarian (SAIC) 

CSTL Program Area: Energy and Environmental Technologies

 

Absorption Coefficient (dimensionless) at 300 K

0.001

0.65

0.40

0.02 0.04

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

QF (clean) Lab Air on

QF

Soot on QF Carbon

Sphere

Carbon Disk

LDTR Experiments

Greenhouse effects associated with climate

change may be influenced strongly by the chemical 

and physical properties of aerosol particles, i.e.,

particulate matter (PM), in the atmosphere. 

Although a variety of methods are used to measure 

atmospheric aerosol black carbon mass, those 

based on thermal-optical analysis (TOA) are widely 

used in the US. TOA methodology makes critical 

and untested assumptions about the thermal and optical behavior of PM on a quartz fiber

collection substrate, as well as the instrument-produced byproducts of pyrolysis. In response, we

have developed a new approach to

characterizing the optical behavior of 

soot PM, which combines a laserdriven thermal reactor (LDTR), an

acoustic levitator, and Stokes/antiStokes Raman spectroscopy as a

well-controlled thermal environment 

for the non-intrusive determination of

thermal-physical and chemical-kinetic 

properties. 

Currently, the largest uncertainty in 

predicting the change in Earth’s 

global average temperatures over 

time is due to inadequate knowledge 

of the optical properties of 

atmospheric aerosols such as soot 

and cloud condensation nuclei. 

LDTR measurements were performed to determine the 

effect of carbon shape and size on its absorption 

characteristics. Results were obtained for a spherical and 

disk-shape agglomeration of compacted particles. This 

information is important for characterizing particles when

their surface is not always spherical and well-predicted by 

theory. Measurements were carried out for different 

average particle temperatures, up to 1200 K, to determine

the heat transfer characteristics of cleaned and baked

quartz fiber filters (QF), which serve as the substrate for 

PM collection. Using a direct laser heating approach, 

absorption coefficient results were obtained at different 

sample temperatures (from ambient to 1180 K) and gas 

pressures (at 75 Torr and 750 Torr) for cleaned filters,

Levitated 

polystyrene

sphere 

Copper 

sphere 

Acoustic

levitator 

transmitter

187
indoor air particles, and soot from an acetylene-fuel flame (above figure). It was observed that the

absorption coefficient increased with temperature for the cleaned filters, whereas the absorption 

coefficient decreased for the filter exposed to indoor air particles and soot. Significant progress 

was made in integrating Raman spectroscopy diagnostics and the acoustic levitator, which is

designed to circumvent the use of a substrate for particles in the LDTR. New reactor copper 

spheres were fabricated to allow for levitation of soot particles inside the sphere, and to provide 

optical access for the Raman measurements (figure above).

Next Steps: We plan to use this technique to develop a unique database that includes, in

addition to soot absorption coefficient, other optical and physical properties for soot, other 

representative samples of particulate matter, and multiphase and multicomponent liquid droplets

that are representative of cloud condensation nuclei characteristics. These data will be used to

provide input information for climate change models, and to improve the performance of optically 

based devices used to monitor particulate matter in the environment. 

Mapping the Protective Antigen Binding Site on the Anthrax Toxin Receptor 

Kevin D Ridge (831) 

CSTL Program Area: Forensics and Homeland Security 

Anthrax is a disease caused by the gram-positive, spore forming bacterium Bacillus anthracis. 

Humans are typically incidental hosts through ingestion of

contaminated foods of animal origin, various animal 

products, and more recently, malicious exposure. 

Because of the heightened public awareness and

concern surrounding anthrax, there is an acute need to

understand the molecular mechanisms governing the

intoxication process and to apply this knowledge to the

development of new detection methods and therapeutic 

strategies that confront this disease. The major 

contributing factor to the pathogenesis of Bacillus anthracis is a secreted, three-component toxin.

The three proteins comprising the toxin are the lethal factor (LF), the edema factor (EF), and the

protective antigen (PA). Although the mechanism of anthrax intoxication is not entirely clear, some 

discrete steps have been delineated through a variety of experimental approaches. Initially, PA

binds to a cell surface integral membrane protein, the anthrax toxin receptor (ATR), and is

cleaved by a cell surface protease generating a truncated carboxyl-terminal form of the protein

(PA63). PA63 possesses the inherent ability to self assemble and the oligomeric form of the

protein is able to bind EF or LF with high affinity. The resulting complexes are then internalized

where they exert their toxic effects. 

CSTL researchers are 

unraveling the mysteries of 

anthrax intoxication to allow 

for early detection and 

subsequent intervention 

protocols responding to toxin 

exposure.

A comprehensive understanding of the molecular mechanisms 

governing the pathogenesis of Bacillus anthracis requires quantitative

information about the interaction of PA with its host cell membrane

bound receptor, ATR. As the binding of PA to ATR can be considered

as the first step in the intoxication process, it represents a potentially 

important target site for early detection and therapeutic intervention.

However, since little is currently known about this interaction at the

molecular level, mapping of the PA binding site on ATR is of intense 

interest to the scientific community as well as those charged with 

protecting public health. Mapping the PA binding site(s) on ATR is

necessary for advancing our understanding of these biological 

macromolecules and their mechanistic role in the pathogenesis of

Bacillus anthracis. Clearly, knowledge of the critical “establishment” 

stages of anthrax intoxication should provide decisive targets for the

early detection of anthrax and subsequent intervention protocols responding to toxin exposure. 

Bacillus anthracis, 

protective antigen 

(PA) Source: PDB 

188
Next Steps: The availability of the ATR gene enables

a systematic study of the PA binding site. Edward

Eisenstein of UMBI/CARB, who is investigating PA63 

assembly, has constructed a synthetic PA gene. With 

these proteins in hand, detailed biochemical and

biophysical measurements of the ATR/PA interaction

are likely to provide a quantitative description of the

anthrax intoxication process 

Mapping the Protective Antigen (PA) 

binding site(s) on the Anthrax Toxin 

Receptor (ATR) is necessary for 

advancing our understanding of 

these biological macromolecules 

and their role in the pathogenesis of 

Bacillus anthracis. 

Development Of SEM-EDS Analysis Procedures For C, N And O To Enable 

Characterization Of Explosives Particles, Polymeric And Organic Materials

J.T. Armstrong and J.A. Small 

CSTL Program Area: Forensics and Homeland Security

Procedures were developed for quantitative phase identification of organic particles and surface

layers using energy dispersive x-ray analysis (EDS) in a scanning electron microscope (SEM) to

determine C, N, and O relative concentrations as 

well as those of heavier elements. Tests of these

procedures on gunpowder grains and explosives 

microparticles showed that, although radiation

damage induces partial dehydration in these

materials, repeatable steady-state residual 

compositions are left that can be used for accurate

phase identification. 

CSTL scientists have determined that 

accurate EDS analysis or phase 

fingerprinting of C, N, and O will open 

a vast potential range of applications 

for analytical scanning electron 

microscopy in many areas. 

Using several commercial thin window EDS analyzers, we found that C, N, and O can be

routinely measured with detection limits of better than 0.5 % for C and O and ~1 % for N in the 

presence of major C. Measurement precisions of ~0.5 % (absolute) can be attained for these 

elements. Although some commercial software packages were found to fail in attempts to

separate peaks from backgrounds for these elements, sequential simplex in NIST's DTSA 

program was found to work satisfactorily. Optimum correction procedures for these elements

were identified and tested on standard (inorganic) multilayer specimens (insulating organic 

particles need to be coated with a conducting material, we chose Au). Results showed 

comparable accuracy to that obtained for elements like Na, Mg, Ga, Ge, and As (commonly 

analyzed elements having low energy x-ray lines). The major problem found in analyzing organic 

materials was, as expected, radiation damage and decomposition under electron bombardment.

Procedures were tested that minimized this damage, but none were found so far that completely 

eliminate it. We found, however, that the degree of decomposition during analysis could be

controlled, resulting in repeatable, distinctive residue compositions – even for materials as volatile

as RDX explosive. The data suggest that by use of controlled conditions, “fingerprint” C-N-O EDS 

spectra can be obtained from analysis of organic particles and thin layers that could be used to

identify their initial phase composition. (See figures 1 and 2 on the next page.) 

Procedures were tested that 

minimized the radiation 

damage and decomposition 

under electron bombardment 

expected when analyzing 

organic materials.

Accurate EDS analysis or phase fingerprinting of C, N, and O 

will open a vast potential range of applications for analytical 

scanning electron microscopy that could potentially double its 

usage or more in areas as important and disparate as forensics 

and homeland security, nanotechnology, microelectronics,

pharmaceutical analysis, corrosion research, failure analysis, 

and environmental sample analysis. 

189
0.0

0.2

0.4

0.6

0.8

0 100 200 300 400 500

e- bombardment (sec)

N/C & O/C Intensity ratio

I-N / I-C

I-O / I-C

RDX Particles

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 200 300 400 500 600

e- bombardment (sec)

N/C & O/C Intensity ratio

I-N / I-C

I-O / I-C

N/C scan

O/C scan

a Winchester Gunpowder b

Å 150 μm Æ

Point 2 Æ

Å Point 1

Å Scan

2500x

Å Scan

1000x

Å Scan 300x Æ

Gunpowder

Particle 2

Å 150 μm Æ

Point 2 Æ

Å Point 1

Å Scan

2500x

Å Scan

1000x

Å Scan 300x Æ

Gunpowder

Particle 2

Gunpowder

Particle 1

 Scan 

2500x

Point 1

60 μm 



Fig. 2. SEM images of 

gunpowder particles. 

Note visible radiation 

damage inflicted by the 

electron beam during 

analysis. Minimal 

damage is seen for the 

‘whole particle’ 300x 

scan in Fig. 2b; 

however, even here 

significant oxygen loss 

occurred (Fig. 1b). 

Fig. 1. Decrease in relative intensities of nitrogen and oxygen with respect to carbon as a 

function of electron bombardment time for RDX (a) and gunpowder (b). Trends are determined 

by a series of replicate analyses in the same spot. In both materials, a distinctive steady state 

composition of residual C-N-O is obtained. 

Next Steps: We propose ramping up this project to develop experience with a wider range of

organic materials and a wider range of beam dose conditions. We also propose testing whether 

cryoanalysis would significantly improve sample stability measurement reproducibility. 

DNA Damage by the Antitumor Drug 3-Amino-1,2,4-benzotriazine 1,4-dioxide 

(Tirapazamine) 

M. Birincioglu, H. Rodriguez, M. Dizdar (831); P. Jaruga (UMBC, MD); and G. Chowdhury

and K. Gates (University of Missouri) 

CSTL Program Area: Health and Medical Technologies

Hypoxia is a typical feature of cancerous tumors. Drugs 

activated in hypoxia have wide potential applications, 

particularly in overcoming the resistance of hypoxic tumor

cells to chemotherapy. Key features of such drugs include

redox properties activated by reductase enzymes such as 

flavoproteins. In hypoxic tumor cells, the enzymatically reduced drug and/or its intermediates lead

to formation of reactive hydroxyl radicals that effectively damage DNA of tumor cells, resulting in

destruction of tumors. Tirapazamine is such a bioreductively activated DNA-damaging agent that

selectively kills the hypoxic cells found in solid tumors. 

CSTL results provide 

researchers with expertise 

in the measurement of 

oxidative DNA damage. 

This compound shows great clinical promise and is currently being examined in a variety of

clinical trials, including several phase III studies. It is well established that DNA is an important

cellular target for tirapazamine; however, the structural nature of the DNA damage inflicted by this 

190
drug remains poorly understood. As part of an effort to understand the chemical events

responsible for the hypoxia-selective cytotoxicity of tirapazamine, the studies reported here were

designed to characterize this drug's ability to damage DNA. Here,

we used gas chromatography/mass spectrometry and liquid

chromatography/mass spectrometry to characterize and quantify 

oxidative DNA damage mediated by tirapazamine. A multiplicity of

modified DNA bases were identified and quantified. It appears that

the hypoxic conditions, under which tirapazamine operates, along 

with the unique chemical properties of the drug, yield a unique

variety of DNA damage that includes poorly repaired, potentially 

cytotoxic DNA base lesions that block DNA transcription and replication. Overall, the results 

indicate that DNA base damage may contribute to the biological effects of 

tirapazamine in vivo. The results also provide researchers in industry and 

academia with expertise in the measurement of oxidative DNA damage, this 

work supports the efforts to develop methodologies to test new anticancer 

drugs similar in chemical structure to tirapazamine and to help elucidate the 

mechanism of action of hypoxia-selective antitumor drugs and their efficacy to

destroy tumor cells, but not normal cells. 

This work provides the 

first detailed insight 

regarding the structural 

identity and quantities of 

the DNA lesions caused by

this new anticancer drug.

Tirapazamine

Next Steps: This study will be extended to the characterization of DNA damage by this drug in

vivo, and by other structurally similar hypoxia-selective antitumor drugs.

Human Mitochondrial Protein Database: A Resource for Human Mitochondrial Proteomics 

V. Ravichandran, P. E. Barker, G. B. Vasquez, T.N. Bhat, G.L. Gilliland (831); S. J. Zull (ATP) 

CSTL Program Area: Health and Medical Technologies

A publicly available web-based resource, the

Human Mitochondrial Protein Database (HMPD), 

has been developed to unify data from a broad 

collection of related resources that provides 

information about human nuclear and 

mitochondrial-encoded proteins. Our database is 

designed to address the problem presented by 

the increasing amount, complexity, and types of 

proteomics data available in public databases. At this point, no systematically established 

correlations between the different types of data have been established that allows meaningful 

comparisons. One of the primary reasons for this is the lack of data standards. Despite this, a

wealth of data exists and is readily available from numerous web resources that are applicable to

proteomics and have direct relevance to identifying proteins and assigning function. However,

serious problems with the interactions between these resources arise because of problems with 

data exchange and interoperability. Although public proteomics data resources are highly 

informative, individually, the collection of available content would have more utility if provided in a

standard and centralized context and indexed in a robust manner for a specialized area. The

HMPD currently consolidates information from many publicly available sources. 

The Human Mitochondrial Protein 

Database (HMPD), a centralized 

database for mitochondrial proteins, 

has been developed as an instrument 

for standardizing proteomics data 

using the mitochondrion as a model 

system. 

A customizable interface has been developed to permit

complex queries that include the name of the protein, 

tissue, mitochondrial compartment, chromosome number,

molecular weight range, pI range, and keywords. Users 

can also restrict the data being searched to 2-D PAGE 

images, Locus links, the Genome Database, OMIM, or

RefSeq information. To further narrow the search, selected 

results are grouped with gene name, Protein name and

SwissProt ID, where the SwissProt ID is linked with detailed information for a protein of selection.

The HMPD web site, 

http://bioinfo.nist.gov:8080/examp

les/servlets/index.html, was 

recently cited in Science’s 

NetWatch (“RESOURCES: Probing 

the Cell’s Powerhouse,” Science, 

300, 713, 2003). 

191
A graphical tool was developed to 

visualize the human mitochondrial 

DNA sequences (16,569 bp). A 

mouseover feature has been added to 

include the annotated information. In

addition, a polymorphism check tool 

has been implemented to dynamically 

compare the change in mtDNA 

changed between two populations, and 

a standard reference data set for twodimensional gel electrophoresis has 

been implemented. 

Next Steps: Future plans include 

developing this web site as a standard 

reference data for mitochondrial 

proteomics. Experimental data from 

areas that impact proteomics will be archived and incorporated into the database (HMPDB). Data

definitions and standards dictionaries will be developed to facilitate data acquisition, display, and

exchange. 

Silicon Nanoparticles as DNA Labels 

Vytas Reipa and L. Wang (831) 

CSTL Program Area: Health and Medical Technologies 

DNA hybridization assays are increasingly applied for

pharmacogenomic research and drug discovery, infectious and genetic 

disease and cancer diagnostics. Micro analytical devices, highthroughput simultaneous test arrays, capillary electrophoretic analysis 

and dipstick devices have presented new challenges and requirements 

for nucleic acid detection, and fluorescent methods currently dominate

in many of these applications. The continuing quest for improved

labels for these assays has produced a series of candidate labels 

including genes encoding enzymes, quantum dots, and new examples 

of the fluorophore (e.g. cyanine dyes) and enzyme class of labels. 

Organic fluorophores, either covalently linked or intercalated to DNA 

are subject to photo degradation, environmental sensitivity, which may 

limit their widespread application in high light intensity methodologies,

such as DNA micro arrays. Manufacturing of such chips also requires 

rigorous quantitation of the amount of surface attached oligonucleotides, which is currently 

problematic using intercalating organic dyes. We explored the applicability of highly luminescent

silicon nanoparticles for DNA tagging in aqueous solutions. The silicon nanoparticles possess 

superior properties, such as high photo-stability and low toxicity, which are crucial for applications 

of luminescence (fluorescence)-based assays in clinical diagnosis. 

1 nm diameter 

Hydrogen capped 

Si particle 

(Biophotonics 

International).

The multiple chemical steps for 

obtaining oligonucleotideconjugated silicon 

nanoparticles include an 

electrochemical etching step

and two photo-induced reaction

steps followed by a DNA 

labeling step that allows the

formation of a carboxamide Oligonucleotide-conjugated silicon nanoparticles

192
bond between silicon nanoparticle and oligonucleotide. The procedure enhances aqueous

solubility of hydrophobic silicon nanoparticles, which is a key requirement for the effective use of

nanoparticles in biological applications. We successfully conjugated 1 to 2 nm diameter silicon

nanoparticles to a 5’-amino modified oligonucleotide whose sequence is from rat tubulin genome.

Under UV excitation, photo-luminescence of the conjugates is dominated by two blue bands (400

nm and 450 nm maximal). The quantum yield of oligonucleotide-conjugated nanoparticles that

were coated with a layer of 1-octene was determined to be 0.08 as measured against quinine

sulfate in 0.1 M HClO4 as a reference standard. We have demonstrated the feasibility of the

present coupling chemistry to obtain silicon nanoparticle-labeled nucleotides. 

It is expected that the alternatives will be superior in the 

following respects: high luminescence quantum yield, high

photo-stability, low toxicity, and size-dependent

luminescence. These properties are critically important for

biological applications such as DNA sequencing, microarrays 

for high-throughput screening, flow cytometry measurements,

and fluorescence in-situ hybridization. 

The research will facilitate the 

development of alternatives to 

organic fluorophores which 

will provide superior 

performance when used in

biological applications.

Next Steps: Size exclusion chromatography separation and the use of a longer carbon chain will 

likely enhance the luminescence quantum efficiency of the nanoparticles, which is critical for 

applications such as fluorescence in-situ hybridization in chromosomes. 

Predicting Vibrational Spectra of Dissolved Salts 

LiLi Wang (831); K. K. Irikura (838) 

CSTL Program Area: Pharmaceuticals and Biomanufacturing

Aqueous ions are commonplace and important

in biology, geochemistry, many manufacturing

processes, and elsewhere. Vibrational 

spectroscopy (infrared, Raman, and nearinfrared) is often used to analyze or monitor 

such systems. For example, most biological 

molecules are ions, and vibrational spectroscopy

is important for identifying their structures and

interactions. For chemical analysis, molecules 

can only be identified by spectroscopy if the 

spectra of candidate molecules can be predicted in some way, for comparison. Modern

theoretical methods can now predict gas-phase spectra with useful precision, but predicting

condensed-phase spectra is more challenging. Ions are especially difficult because they interact 

strongly with the solvent or matrix, often causing dramatic changes in the spectrum. Although 

significant progress has been achieved in modeling solvation, the

primary emphasis has been on energetics, with a secondary emphasis

on molecular structure. The performance of solvation models in 

predicting vibrational spectra using quantum chemistry has received

little attention. 

CSTL scientists use vibrational 

spectroscopy to identify compounds by 

comparing observed spectra with those 

of candidate compounds. Despite the 

ubiquity of ions throughout chemistry, 

to date there are no reliable theoretical 

procedures for predicting their 

vibrational spectra in solution. 

To date, the most popular, inexpensive continuum models have been

found inadequate, and have failed rigorous testing. Consequently,

computations with explicit solvent molecules are now underway. Two

reasonable electronic-structure models have been identified and

compared for the prototype ion H2O+

 solvated by neon, which was 

chosen as the initial, simple case for which benchmark experimental 

data are available. Increasingly large computations still have not 

reached the bulk limit. 

A sample structure 

for H2O+

(Ne)13 

193
Next Steps: Ion-solvent clusters are mechanically “soft”, with

multiple, competing structures, and the corresponding effect upon

the vibrational spectrum remains to be investigated. Simulatedannealing approaches will also be applied to find the most stable 

structure for each cluster. Both procedures will help in reaching 

the bulk limit, a major goal of this project.

Reliable theory will permit 

observed spectra to be 

assigned with confidence, 

thus providing the 

necessary interpretation of 

the experimental data. The 

current limited success is

essential for aqueous 

frequency predictions. 

Trace Nanoanalysis Using Hyperspectral Imaging 

J. H.J. Scott and J. M.Conny (837) 

CSTL Program Area: Technology for Future Measurement

The recent introduction of multispectral and

hyperspectral imaging techniques to analytical 

electron microscopy and electron probe 

microanalysis has led to an explosion in the

amount of chemical data available from each 

sample. Thousands of channels of spectral

information can be saved for each of millions of

pixels on the specimen, and the challenge is to

find techniques that can exploit this wealth of

raw data. Following the lead of analytical 

chemists working with non-microscopic 

methods, this work attempts to apply 

multivariate statistical tools and chemometrics to meet this challenge. The goal is to provide a

new suite of analysis tools for the microanalysts in government labs, academia, and the industrial 

sector. The NIST/NIH Desktop Spectrum Analyzer (DTSA) used for testing new statistical 

approaches and validating methods such as Principal Components Regression (PCR) and Partial 

Least Squares (PLS) for multivariate calibration. Software implementing these algorithms (in

Matlab) was linked to DTSA output via a series of scripts. This allows ideas borrowed from 

chemometrics (such as multivariate curve resolution, multivariate image analysis, and inverse 

calibration methods) to be applied to current problems in microanalysis.

CSTL Researchers develop tools and 

utilities for exploring the application of 

chemometrics to trace nanoanalysis in 

the analytical electron microscope 

(AEM) and electron microprobe; and 

apply multivariate data analysis 

strategies developed for bulk analytical 

methods to chemical analysis at high 

spatial resolution in the analytical 

electron microscope.

In FY 03, these researchers accomplished the following: 

• Implemented a set of scripts for pipelining data from the NIST/NIH Desktop Spectrum 

Analyzer package (DTSA) to the PLS toolbox in Matlab for multivariate analysis 

• Developed an environment for testing the viability of existing methods in chemometrics in 

the context of hyperspectral data acquisition in the AEM and electron microprobe 

• Imported synthetic Fe-Ni-Cr spectral data from DTSA into Matlab to evaluate Principal

Components Regression (PCR) and Partial Least Squares (PLS) as potential methods for

multivariate quantitative microanalysis 

• Imported synthetic datasets for exploring non-linear spectral pre-treatments to optimize

multivariate calibration using energy-dispersive x-ray (EDS) data 

• Used multivariate curve resolution to resolve mixed experimental spectra into “pure 

components” without a priori chemical knowledge of the system; investigated numerical 

convergence problems 

These tools and utilities may permit 

leveraging of existing expertise in 

chemometrics to enhance performance of 

chemical measurement tools such as

energy-dispersive x-ray spectrometry. 

Next Steps: Researchers plan to investigate 

current topics being explored in analytical 

chemistry, chemometrics, and multivariate analysis

and apply them to problems of ultramicroanalysis.

194
Growth of Oriented ZnO Nanowires on Sapphire and Gallium Nitride 

Babak Nikoobakht and Stephan J. Stranick (837) 

CSTL Program Area: Technologies for Future Measurement

Nano-light sources and detectors can be 

realized by engineering a nanowire with a diode

junction/functionality. This along with other

nano-device functionality could enable the

development of sensor arrays capable of 

performing quantitative chemical 

measurements. These methodologies, when

further refined, could be employed to generate

novel opto-electronic devices for use in sensor

sapphire and GaN surfaces via a vapor-phase transport 

process using Au thin film as catalyst. We have been able

to successfully control the growth direction of individual 

ZnO NWs and the morphology of their ensemble. We

present the first examples of vertical and 60o-tilted NWs on

c-plane sapphire, vertical NWs on GaN substrate and 

unidirectional growth of horizontal NWs on a-plane 

sapphire. In the case of normal NWs on sapphire and

GaN, XRD analysis shows that single-crystal wurtzite NWs 

grow in the [0001] direction normal to the basal c-plane,

which proves that a-plane sapphire is not essential for

growth of vertical ZnO NWs. We have found that the

thickness of the Au-film and pre-growth annealing of

Au/sapphire substrate are critical in controlling the 

morphology of the nanowires. Utilizing the above factors, 

two new morphologies have been selectively synthesized.

AFM and SEM studies show that in the absence of 

annealing, Au films with feature heights between 1 to 10

nm result in NWs 60o tilted with respect to the surface,

whereas pre-annealed Au films with heights below ~6 nm

result in growth of NWs in the surface normal direction. For the annealed Au films thicker than 6 

± 2 nm, a network of ZnO is formed followed by the growth of NWs in the surface normal

direction. We have attributed the normal and tilted growth directions to the surface concentration

of O and Al ions on sapphire. In order to form horizontal NWs, results show that the substrate 

needs to be a-plane and instead of an Au

film as catalyst, scattered Au droplets with 

diameter less than 15 nm must be present 

on the surface. In this case NWs all grow

in (110) direction on the basal plane of the

a-plane sapphire. The findings of this

study have been submitted for publication 

in Advanced Materials and Journal of 

Physical Chemistry B. 

Next Steps: Our pre

CSTL researchers develop novel 

methods to grow and manipulate 

semiconductor nanowires that have 

unique properties and make them well 

suited for applications in optoelectronics and as potential building 

blocks of future measurement platforms. 

and proximal probe applications. In FY 03, CSTL scientists grew ZnO nanowires (NWs) on 

liminary studies have focused on the growth and manipulation of

emiconductor nanowire. These methodologies will be further refined and then employed to

500 nm

b

1 μ

a

5 μ

c

1 

e

d

μ

1μ

b

500 nm

b

1 μ

a

5 μ

c

1 

e

d

μ

1μ

b

Electron micrograph of a) and b)

normal NWs on c-plane sapphire and

GaN c) 60 ̊ tilted NWs on c-plane

sapphire d) NWs on ZnO network

(top view) e) horizontal NWs on aplane sapphire 1μm. 

Nanowire devices will provide multiple options as 

building blocks for sensors, miniaturized 

measurement platforms, and as advanced scatter

probe structures, enabling true nanometer 

spectroscopy. This methodology will provide an 

inexpensive and straightforward approach to the 

synthesis of sensing devices with multiple 

functionalities that can be integrated to form

state-of-the-art measurement systems. 

s

generate novel opto-electronic devices for use in sensor and proximal probe applications. 

195
Progress Towards Development of an Improved Method of Phase Identification by 

Selected Area Electron Diffraction

S. Turner, D.S. Bright (837); and V.L. Karen, (MSEL) 

CSTL Program Area: Technologies for Future Measurement

Selected area electron diffraction (SAED) is a very

useful technique for identification of materials examined

by transmission electron microscopy. In combination 

with chemical information, the structural data obtained 

by SAED greatly narrows the range of possible phases.

SAED has been limited, however, in that information is 

generally obtained for the sample in only one 

orientation. Currently, if SAED is obtained for more

than one orientation its use is limited because it has 

been difficult to relate the 3-D information in the 

diffraction patterns. The recent availability of a double-tilt, rotate sample holder with improved

manipulation capabilities along with the availability of reduced cell and phase identification 

programs developed at NIST suggested the possibility of an improved method of phase 

identification. Ideally, structural data collected from more than one orientation could be obtained, 

the geometric relationships determined, reduced unit cells derived and the phase identified from

the reduced cells. In this work, the feasibility of this approach was investigated. 

CSTL researchers explore the 

possibility of improved phase 

identification by transmission 

electron microscopy by applying 

electron diffraction data obtained 

from a double-tilt, rotate sample 

holder to reduced cell and phase 

matching programs. 

In FY 03, initial steps were made to interface the electron microscope diffraction data from 

multiple orientations with a reduced cell program for phase identification. Over twenty diffraction

patterns and their angular relationships were obtained from a single tetragonal crystal. Diffraction

patterns were calculated for the material and a comparison of the calculated and experimental

interzone angles showed good agreement. Additionally, diffraction patterns for a cubic material 

were modeled. Application of the acquired and modeled data to the reduced cell program 

resulted in unexpected challenges. We have solved some of the problems and can now generate

reduced volumes that in some cases are consistent with identification of the analyzed phase. We

will continue to address problems in the interfacing of multiple diffraction patterns with a reduced

cell and the associated phase identification program. More work needs to be done to overcome

problems associated with interfacing the experimental data to the programs and to evaluate the

effect of uncertainty in the data on phase determination. 

Next Steps: Work will continue on efforts to address problems in the interfacing of multiple

diffraction patterns with a reduced cell and the associated phase identification program. More

work needs to be done to overcome problems associated with interfacing the experimental data

to the programs and to evaluate the effect of uncertainty in the data on phase determination. 

Universal Validation Standard for UV/visible Spectrometry 

D.L. Duewer and J.C. Travis 

CSTL Program Area: Technologies for Future Measurement 

The traditional suite of UV/visible absorption reference materials designed for research grade 

instruments has fallen short of meeting the traceability requirements of quality systems 

associated with “factory floor” instruments. The goal of this proposal was to develop a universal 

standard that would serve as a replacement for the entire suite of current NIST SRMs (Standard 

Reference Materials) for chemical spectrometry in the UV/visible spectral region. This would be

possible due to the development of a novel whole-spectrum fitting algorithm. This algorithm

provides the core functionality for establishing the UV/visible absorption spectra of holmium oxide

solution (such as NIST SRM 2034) as an intrinsic standard for the total performance validation of 

chemical spectrophotometers. 

196
The declaration of dilute holmium oxide solution as an intrinsic wavelength standard was

underway at the time the proposal was developed, and is reported elsewhere. Data from the 

study that declared dilute holmium oxide solution as an intrinsic wavelength standard were used

in the successful development of an algorithm to fit the entire (certified) intrinsic spectrum to the 

spectrum of properly prepared holmium oxide solution taken on an instrument to be validated. 

The algorithm accounts for the wavelength bias, the absorbance bias, and – most significantly –

the spectral bandwidth of the recording instrument. A significant breakthrough in algorithm

development was the approach by Statistical Engineering Division collaborators to perform a fit 

that actually extracts the experimental instrument function (“slit function”) in addition to the other 

expected bias data. 

The universal standard will provide simultaneous wavelength 

calibration and validation of the absorbance scale and spectral 

bandwidth specification for practical instruments with a single

artifact, data acquisition, and automated data analysis. In

addition to making life easier for the end user, traceability 

support will be almost entirely outsourced from NIST. The

attention of CSTL can then be turned to emerging 

measurement technologies while this mature technology is 

supported by the intrinsic standard. 

The universal standard will 

provide simultaneous 

wavelength calibration and 

validation of the absorbance 

scale and spectral bandwidth 

specification for practical

instruments with a single

artifact, data acquisition, and 

automated data analysis. 

Next Steps: Work is continuing on the certification of 4% (w/v) holmium oxide dissolved in 10%

(v/v) perchloric acid as an intrinsic wavelength standard. With modest additional investment of

resources, we will demonstrate that this material is suitable for use as an intrinsic standard for 

both wavelength and absorbance when spectral baseline artifacts (arising from differences in the 

surface reflectance of individual cuvettes and minor organic contamination of solvents and 

cuvettes) are eliminated experimentally. However, we believe that it is possible to mathematically 

account for these artifacts within the spectral-fit software. This algorithmic development would 

greatly simplify the necessary experimental burden on the end-user and would enable routine use

of the intrinsic standard to meet most chemical traceability requirements. 

197
198
D. Staff Recognition 

Our staff is CSTL’s greatest resource. We are proud of all of them and their accomplishments, both

at work and in their communities. In FY 2003, the following staff were formally recognized for their 

achievements. 

ir work with

tering of

ted in the

ternational

semiconductor industry’s chip fabrication lines. 

John Butler (831) received the Scientific Prize from the International Society of

Forensic Genetics for his contributions to the field of rapid and accurate DNA typing

for forensics and human identification. 

Rich Cavanagh (837) was elected a Fellow of the American Vacuum Society for 

his sustained and outstanding contributions in areas of interest to the AVS. 

Bill Dorko (839) received the Department of Commerce Silver Medal for the 

development and implementation of the NIST Traceable Reference Materials (NTRM) 

Program for gas mixture standards. 

Carol Driver (838) received the CSTL Secretarial Achievement Award for her 

outstanding skills and providing extraordinary service to the staff and programs of

CSTL. 

David L. Duewer (839) received the Edward Bennett Rosa Award for his innovative

use of visualization techniques to summarize and communicate complex statistical 

information. The Edward Bennett Rosa Award is granted for outstanding

achievement in or contributions to the development of meaningful and significant 

engineering, scientific, or documentary standards.

John J. Hurly and Keith A. Gillis (836) were

Department of Commerce Bronze Medal for the

semiconductor process design and the me

semiconductor process gases, needs documen

1999 Semiconductor Industry Association (SIA) In

Technology Roadmap for Semiconductors. 

awarded the

Robert Greenberg, Richard Lindstrom (839) and David

Simons (837) received the Judson C. French Award for their 

development and critical evaluation of an instrumental

neutron activation analysis method for the determination of

arsenic in silicon and transferring this technology to the

199
Frank Guenther (839) received the Department of Commerce Silver Medal for the 

Donna Kimball (839) received the Eugene Casson Crittenden Award for her 

ick Lindstrom (839) received the Department of Commerce Silver Medal for the

ation, health care, and high performance electronics.

development and implementation of the NIST Traceable Reference Materials 

(NTRM) Program for gas mixture standards. He was also awarded the Safety 

Award for Superior Accomplishment for the development and implementation of an

efficient process for inventorying and cataloging chemicals used within the

Analytical Chemistry Division of CSTL. 

continued energy, leadership skills, and organizational talents which she used to coorganize and coordinate the NIST “Take Our Sons and Daughters to Work Day”

Program. She also managed all logistical aspects of the NIST Centennial 

Symposium at the 2001 Pittsburgh Conference. 

D

development and delivery of cutting-edge nuclear technology to support U.S.

nonproliferation efforts. 

Gerald Mitchell (839) was awarded the Safety Award for Superior Accomplishment

for the development and implementation of an efficient process for inventorying and

cataloging chemicals used within the Analytical Chemistry Division of CSTL.

Ray Radebaugh (838) has been honored with the Department of Commerce Gold

Medal for his outstanding contributions to the successful transfer of cryocooler 

technology to U.S. industry for a broad range of applications including military 

surveillance, space explor

David Ross (836) was named the Sigma Xi Young Scientist of the year for 2002-2003 

for his seminal research in the area of microfluidic systems. 

Jennifer Scott (838) is the winner of the NIST Boulder Building Tomorrow’s 

Workplace Award (BTW) for her leadership role in encouraging mentoring

activities and in establishing a positive environment for recognizing the potential of

strengthening the effectiveness of the organization by making use of the

differences in the workforce personnel 

200
The CSTL Technical Achievement Award went to the team 

responsible for the certification of SRM 1946, “Lake Superior Fish

Tissue.” Michele Schantz and Stephen Wise (839) were the 

project coordinators. Contributing analysts were Wayne Brubaker, Jr., Steven Christopher, John 

Kucklick, Stephen Long, Elizabeth Mackey, Curtis Phinney, Barbara Porter, Dianne Poster,

Rebecca Pugh, Michael Rearick, Katherine Sharpless, and Silke Tutschku. 

SCO.

vative, chemoe, or drift, that

y up to 20-fold. 

 (836) received the Department of Commerce Bronze Medal for

Anneke Sengers, scientist emeritus, was selected as the North American recipient

of the 2003 Women in Science Awards presented by the cosmetics company

L’Oreal and the international organization UNE

Steve Stranick (837) received the ACS Analytical Chemistry Division Arthur F.

Findeis Award for his contributions to the fields of analytical chemistry including his

development of a novel microscope that combines the high spatial resolution of

near-field scanning optical microscopy with the quantitative chemical probe

capabilities of infrared spectroscopy. 

Wing Tsang (838) was appointed to the position of NIST Fellow. Dr. Tsang is

recognized for his world-class accomplishments and leadership in the area of

chemical kinetics.

Marc Salit and Greg Turk (839) were awarded the

Commerce Bronze Medal for for their work on an inno

metric-based approach to correct for low frequency nois

has improved the precision of chemical measurements b

Department of

John D. Wright

designing, assembling, testing, and publishing a definitive description of an

extraordinary, new gas-flow standard. 

201
202
The CSTL Divisions 

CSTL’s laboratory activities are primarily located at the NIST headquarters site in Gaithersburg, MD. We

also have research activities in NIST laboratories in Boulder, CO, and at the Center for Advanced Research 

in Biotechnology (CARB) in Rockville, MD. CARB is a collaborative effort of NIST, the University of 

Maryland, and Montgomery County, MD. 

Chemical Science and Technology Laboratory

Dr. William F. Koch, Acting Director

(301) 975-8301 

william.koch@nist.gov 

Eric Steel, Acting Deputy Director

(301) 975-8300 

eric.steel@nist.gov 

Biotechnology Division

Dr. Vince L. Vilker, Chief 

(301) 975-2629 

vincent.vilker@nist.gov 

3⁄4 DNA Technologies 

3⁄4 Bioprocess 

3⁄4 Structural Biology 

3⁄4 Biomolecular Materials 

Process Measurements Division

Dr. James R. Whetstone, Chief 

(301) 975-2600 

james.whetstone@nist.gov 

3⁄4 Fluid Flow

3⁄4 Fluid Science

3⁄4 Process Sensing

3⁄4 Thermometry

3⁄4 Pressure and Vacuum

3⁄4 Thermal and Reactive Processes

Surface and Microanalysis Science Division

Dr. Richard R. Cavanagh, Chief 

(301) 975-2368 

richard.cavanagh@nist.gov 

3⁄4 Microanalysis Research 

3⁄4 Surface and Interface Research 

3⁄4 Analytical Microscopy 

Physical and Chemical Properties Division

Dr. Gregory J. Rosasco, Chief 

(301) 975-2483 

gregory.rosasco@nist.gov 

3⁄4 Experimental Kinetics and Thermodynamics 

3⁄4 Chemical Reference Data and Modeling 

3⁄4 Computational Chemistry 

3⁄4 Experimental Properties of Fluids 

3⁄4 Cryogenic Technologies 

Analytical Chemistry Division

Dr. Willie E. May, Chief 

(301) 975-3108 

willie.may@nist.gov 

3⁄4 Spectrochemical Methods 

3⁄4 Organic Analytical Methods 

3⁄4 Gas Metrology and Classical Methods 

3⁄4 Molecular Spectroscopy and Microfluidic Methods 

3⁄4 Nuclear Methods

Visit the CSTL Website at: 

http://www.cstl.nist.gov/ 
 February 2004