Chemical Science and Technology Laboratory 

Annual Report – FY2004 

NISTIR 7202
NISTIR 7202 

Chemical Science and Technology Laboratory 

Annual Report - FY2004 

Willie E. May, Director 

William F. Koch, Deputy Director 

Chemical Science and Technology Laboratory 

February 2005 

U. S. DEPARTMENT OF COMMERCE 

Carlos Gutierrez, Secretary 

TECHNOLOGY ADMINISTRATION 

Philip J. Bond, Under Secretary for Technology 

NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY 

Hratch G. Semerjian, Acting Director 
Compiled and Edited by Ellyn S. Beary and Pedro Espina. 

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 2005 
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 21 

 3. Pharmaceuticals and Biomanufacturing 25 

 4. Chemical and Allied Products 29 

 5. Energy and Environmental Technologies 41 

 6. Food and Nutritional Products 57 

 7. Forensics and Homeland Security 59 

 8. Health and Medical Technologies 75 

 9. Industrial and Analytical Instruments and Services 91 

 10. Microelectronics 111 

 11. Measurement Standards 121 

 12. Data and Informatics 133 

 13. Technologies for Future Measurements and Standards 143 

C. Exploratory Research 157 

D. Staff Recognition 167 
ii
iii

A Message from the CSTL Director 

The Chemical Science and Technology Laboratory is one of seven discipline-based 

technical operating units at the National Institute of Standards and Technology. All of 

us in CSTL are proud to share with you our Annual Report for Fiscal Year 2004. While 

this report represents only a snapshot of our activities, it illustrates the wide breadth of 

research that we conduct and measurement services that we provide. 

During this past fiscal year, Dr. William F. Koch served as Acting Director and ably 

led CSTL through an aggressive strategic planning process. He began serving in this 

capacity in July 2003, when Dr. Hratch Semerjian was appointed the Deputy Director 

of NIST. I became Director of CSTL in late December 2004 having served as Chief of 

CSTL’s Analytical Chemistry Division for the past ten years. It is both an honor and a 

privilege to take over the helm, and to advance the creative research activities and delivery of world-class 

measurement services for which CSTL has become internationally renowned. 

This past year has witnessed many changes in our Laboratory. In addition to the leadership changes just mentioned, 

many other changes have occurred, including our move into our new Advanced Measurement Laboratory—a truly 

unique facility with respect to air cleanliness, and temperature, humidity, and vibration control. All proceeded in a 

very smooth manner with no adverse impact on the research and measurement services that CSTL provides. This is 

truly a testament to both the depth of leadership within our Laboratory, and the quality, creativity, and dedication of 

our staff. 

We have been challenged in recent years with relatively flat 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 provide the largest benefits 

to US citizens, and return on investment for our Nation. In this regard, we will continue to be proactive in 

communicating the breadth and depth of our capabilities as well as the quality and impact of our work. We will 

continue to strengthen our interactions with industry and other stakeholders in order to help us prioritize our work. We 

are grateful for the candid input and ideas that these colleagues have shared with us in the past and look forward to 

continued fruitful interactions. 

In closing, the NIST Chemical Science and Technology Laboratory has world-class facilities, and the most 

comprehensive array of chemical, physical, and engineering measurement capabilities of any group working in the 

general area of chemical science and technology. We will continue to be well-positioned as “a world-class research 

laboratory 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 US competitiveness in the world 

market and facilitate improved quality of life. While maintaining our focus on NIST’s core mission responsibilities 

for the measurements and standards that we have traditionally supported, we are expanding our programs in 

Bioscience and Health, Nanometrology, Data and Informatics, and Homeland Security. The main body of this report 

reflects the increased focus in these areas and the critical synergistic relationships that exist among our research 

programs in measurement science, and the measurement services that we provide to our customers. 

Willie E. May, Director 

Chemical Science and Technology Laboratory 
iv

Hratch Semerjian, Acting Director 

Richard Kayser, Acting 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 nonregulatory federal agency within the US 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. Technology advances account for up to half of new economic growth. It strengthens 

America's security, improves the health of the American people, and creates new jobs and 

opportunities to support our future prosperity. NIST plays a key role in enabling the hard work 

and innovative ideas of the American people to strengthen our technology leadership. NIST works with the private 

sector to develop and apply the technology, measurements and standards that industry needs for new and improved 

products and services. In FY 2004, NIST had an operating budget of about $771 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.

http://www.nist.gov
1

 

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 US, thereby enhancing US 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 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. 

CSTL first developed its mission, vision and goals statements in 1992, 

and then updated them in 1995 and 1998. While they were clearly 

aligned with the NIST mission and vision, ours were articulated separately. As our thinking in strategic planning has evolved, CSTL now 

refers to its role in NIST’s mission in the following manner: CSTL 

fulfills NIST’s mission by addressing customer needs for measurements, standards, and data in the areas broadly encompassed 

by chemistry, biosciences, and chemical engineering. 

Likewise CSTL looks to the NIST Strategic Plan to identify CSTL’s 

part in its realization. This strategic plan is by its nature visionary, long 

term, and at a high level. It sets a clear direction and course for NIST. 

CSTL, in the context of this plan, identifies, explores, develops, and 

refines those opportunities that lie in the areas of chemistry, chemical 

engineering, and bioscience. CSTL’s strategy and substructure is flexible and fluid enough to respond quickly to changes in National priorities, escalating and changing customer needs, and the rapid evolution 

of science and technology. 

CSTL is working toward a Shared Vision – one that all can understand and embrace. The most current version is simply 

stated: that CSTL is seen by its staff and stakeholders as providing value to our customers with the right measurements, 

standards, and data at the right time. Some measures of success are: we identify our customer needs in an open and 

consistent process; we understand, anticipate, and respond cost-effectively to customer needs; we balance research and 

services, projects and people, and new and existing strengths, skill sets, and expertise; we work collaboratively and in 

partnership to leverage resources for the best strategic and stakeholder outcomes; we advance the fundamental science 

basis for global measurement systems, and we consistently assess the value and impact of our work. 

Director,

 Willie E. May 

Deputy Director, 

 William F. Koch 

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. 
2

As we developed this vision, certain key points emerged: this vision is dynamic – it will become clearer and may shift 

focus over time; it is not about repackaging what we’re already doing; we must develop and/or adapt specific strategies 

and tactics to achieve this vision; not everything or everyone is always going to fit; and communication, transparency 

and teamwork will become norms throughout CSTL in order to foster effective involvement and trust, and enable the 

best ideas to emerge. 

In pursuit of this Vision, CSTL provides technical leadership for the Nation’s measurement and standards infrastructure 

in synchrony with NIST’s Strategic Plan. The Strategic Plan allows us to respond to National priorities, changing customer needs, and the rapid evolution of science and technology. 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 and even enhanced. Our project planning and priority setting are driven by our three goals listed below: 

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 US; and 

• Supporting and strengthening voluntary standards organizations.

Chemical and Process Information: Assure that US 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. 
3

CSTL Organizational Structure, Leadership, 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. 

The discipline-based organizational structure provides a snap shot of core competences and technical expertise, but does 

not adequately describe the dynamic vision of the CSTL operations in the current technology environment that requires 

a multidisciplinary approach that is responsive and agile. This model was developed by the CSTL leadership team in FY 

2004, and is embraced as an operational vision. The key points are that CSTL is comprised of its entire staff from bench 

to technical leaders through senior management. All have a key role to play in making CSTL an organization that is 

seen by all (staff and stakeholders) as providing value to our customers with the right measurements, standards and data 

at the right time. The “point of contact model” is based on the understanding that technical opportunities enter the organization at every level, and then are focused by a champion (division chief, technical leader, senior scientist) drawing 

on his/her own expertise as well as other members of the management team, technical leaders, and experts to address 

the scope of the opportunity, the role for NIST and CSTL, and other cross-disciplinary issues. If the opportunity is a 

high priority based on our criteria, the best team is then assembled across technical divisions to provide the greatest 

impact for the customer. 

This philosophical change in the role of leadership and operations has served us well over the past year. This past 18 

months saw transitions in leadership at all levels culminating in the appointment of CSTL’s new Director, Willie E. 

May, in December of 2004. This resulted in a cascade of leadership changes within the Analytical Chemistry Division 

including the appointment of a new Chief, Stephen A. Wise. The Group Leader position he vacated was filled by Lane 

C. Sander. There were similar changes in the Biotechnology Division as Vincent L.Vilker was detailed to the Center for 

Advanced Research in Biotechnology (CARB) and Robert N. Goldberg became Acting Chief. His position as Group 

Leader was filled by Adolfas Gaigalas. The facility with which CSTL achieved these major changes is a testament to its 

successful succession planning. 
4

To achieve its goals, CSTL maintains an experienced, well-educated professional staff. The full-time permanent staff 

numbered 249 in FY04, in addition to 79 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 technicalsupport staff augments the professional staff. Approximately 80% of our scientists have PhD degrees. 

CSTL’s physical facilities are located at the major NIST sites in 

Gaithersburg, Maryland and Boulder, Colorado. In addition, CSTL 

shares facilities at the Center for Advanced Research in Biotechnology (CARB) with University of Maryland Biotechnology Institute in Rockville, Maryland. The Hollings Marine Laboratory 

(HML) in Charleston, South Carolina is a cooperative research 

facility involving NIST, the National Oceanic and Atmospheric 

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

South Carolina. 

 

All five CSTL divisions have a presence on the Gaithersburg campus. In addition, the Physical and Chemical Properties Division has operations located in Boulder; the Biotechnology 

Division has its structural biology activity located at CARB; and the Analytical Chemistry Division is the primary liaison with HML, having scientist from two of its groups located in Charleston. 

In the last quarter of 2004 we began expanding our efforts in the Hollings Marine Laboratory (HML) to Marine Health 

and Bioscience, and are exploring opportunities for collaboration with NOAA in their major initiative in Oceans and 

Human Health. CSTL’s Biotechnology Division and Physical and Chemical Properties 

Division are part of the expansion, which include new areas of research in protein 

characterization, and the development of critical Mass Spectral Databases. We are 

also increasing our efforts and research in marine genomics and leveraging these efforts through collaboration with the Department of Marine Genomics of the South 

Carolina Department of Natural Resources, one of the HML partners. In addition we 

are looking at future opportunities for collaborations with NOAA that utilize new 

facilities at the HML such as the Biohazard Level 3 laboratories and the new NMR 

laboratory. 

Administrative 

Support (34) 

Technical 

Support (27) 

Chemists 

(149) 

Engineers, Physicists (41)

 Comp. Scientists (35) 

Biologists, 

Biochemists (25) 

Students (17) 

CSTL Staff, FY 2004 CSTL Funding, FY 2004

Other Agency 

17 % 

Competence 

3 % 

ATP 2 %

Research Base 

66 % 

 Measurement Services 

Funding Sources 13 % 

Total = $66.1
5

The Analytical Chemistry and Biotechnology Divisions have now been located in 

the Advanced Chemical Sciences Laboratory (ACSL) for over five years, the first 

new research facility on the NIST Gaithersburg Campus in over thirty years. 

 In March 2004, our long-anticipated move to the Advanced Measurement Laboratory took place. This move involved most of the staff from the Surface and Microanalysis Science Division as well as the Pressure and Vacuum Group in the Process 

Measurements Division. The state-of-the-art instruments moving into the AML have already demonstrated enhanced 

performance due to the superior environmental control. The high-performance Auger instrument that took four years to 

barely attain the required resolution specification of 10 nm in Building 222, easily met these specifications at installation in the AML Building 217. Similarly the Analytical Electron Microscope met a 0.19 nm resolution specification 

easily in the AML. This bodes well for attaining the two-to-five fold improvement in resolution expected from the next-generation instrumentation that is now 

being acquired. We can now confidently say that the AML is truly the premier 

environment for nanoscale measurements. Attention is now being directed to 

improving the facilities 

in Gaithersburg and 

Boulder not located in 

the ACSL and AML. 

CSTL Strategic Directions 

CSTL has, by design, invested in five major areas since 1997: Biotechnology, Nanotechnology, Healthcare, Data and 

Informatics, and International Measurement Standards. These areas of investment have been refined over the last few 

years to meet the challenge of rapidly changing needs in high-impact areas, and to enhance the vibrancy of our core 

competences. These strategic thrusts, listed below, define our unique role and channel our core competencies to address 

existing and anticipated stakeholder needs, while cross-cutting 

organizational lines. 

1. Nanometrology for chemical characterization, device 

characterization, and processes at the sub-micrometer scale. 

2. Biometrology for biochemically- or cell-derived materials, 

devices, or processes. 

3. Properties Information Infrastructure technologies for generation, evaluation, and dissemination of chemical and 

physical property information.

4. Process Metrology for chemical engineering processes 

5. Chemical Metrology for the chemical characterization of 

materials and complex mixtures. 

The relationship to the NIST Strategic Focus Areas (SFAs) is still 

just as clear, and the obvious mapping to our previous thrusts permits us to track our new and continuing investments. 

We have chosen to focus on the metrological aspects of these emerging and changing areas to more clearly describe to 

our staff what specific role CSTL should play, so as not to confuse our customer-aligned programs with our missiondriven thrusts. Homeland Security is not explicitly named in the CSTL thrusts, 

but our role in this vital National priority is clearly a part of all five of our areas 

of emphasis. In addition to this emphasis on measurement science and research, 

CSTL is fully committed to providing the measurement services required by its 

customers; this includes: constantly aligning our efforts with the NIST role in 

the National Measurement System; implementing quality systems to support 

CSTL measurement services; designing and implementing the best and most 

efficient ways to deliver current and next-generation services. 

NIST Strategic Focus Areas: 

• Nanometrology 

• Biosystems and Health 

• Information/Knowledge 

Management 

• Homeland Security 

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. 

Baseline temperature (20 °C) can be controlled to within ±0.25 °C with accuracies up 

to ±0.01 °C 

Baseline humidity of 40% to 45% RH can be

ACSL
6

Selection of new projects for reprogramming and identification of projects to be phased out requires specific criteria. 

The CSTL Management Council uses the following criteria for its decision-making. The first five are aligned and in 

harmony with the five NIST priority-setting criteria. 

1. Industrial Need – The magnitude and immediacy of industrial need is assessed. 

2. Match to Mission – CSTL meets customer needs for measurements, standards, and data in the areas broadly 

encompassed by chemistry, biosciences, and chemical engineering. 

3. Making a Difference – CSTL’s contribution is unique and critical for success. 

4. Nature and Size of Impact – The measure of anticipated impact relative to investment is evaluated (rate of return). 

5. Timely Quality Output – CSTL has the ability to respond in a timely fashion with high-quality output.

6. Science/Technology (S&T) Opportunity – Recent scientific and technological advances present new opportunities for CSTL. 

NIST revisits and refines these criteria from time to time, having recently added an element of “impact path” and “strategic significance of impact” that relate to criterion number 4 and “probability of success” which adds some dimension 

to criterion number 5. CSTL has added a sixth criterion in overall decision-making since we strongly believe that S&T 

opportunity can be a significant, even key, factor. 

To provide our staff with stimulus for innovative ideas and new areas of research, CSTL continues its tradition of oneyear funding for small seed projects that evaluate the feasibility of new technologies and measurement methods. In 

FY2004, CSTL funded 14 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. 

US 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. 
7

CSTL has long utilized a customer-based programmatic 

structure to help focus CSTL projects and activities, as 

well as to provide a forum to articulate the nature and 

impact of our work. This customer focus, combined with 

the agility of our technical staff, permits CSTL to respond 

in a timely manner to changes in measurement needs 

within each industry segment, and 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. This structure 

remains deliberately flexible and modifications are made 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. 

It is also beneficial to articulate CSTL projects by activities that cross-cut industry sectors. Therefore, CSTL has 

three cross-cutting programs that are mission-driven and 

are clearly aligned with CSTL’s three strategic goals. 

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, Standard Reference Materials (SRMs), Standard Reference Data (SRDs), publications, invited talks, patents, licenses, the number of 

companies using our calibration services, the number of institutions who purchase our SRMs in the US and abroad, and 

the number of institutions who purchase or license our 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. 

11. Measurement Standards 

12. Data and Informatics 

13. Measurement Science for Future Standards 

and Technologies 

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
8

Some of the CSTL outputs and interactions in FY2004 are summarized in the following table. 

Selected CSTL Outputs – FY2004 

Div Pubs Talks Committees Seminars Conferences CRADAs Patents 

Issued 

SRMs/RMs SRDs Cals 

830 1 20 12 8 1 0 0 0 0 0 

831 120 152 39 3 22 3 1 8 2 0 

836 65 45 76 13 5 1 0 1 0 682 

837 38 90 80 10 4 4 1 3 5 0 

838 86 114 83 21 5 6 0 2 16 0 

839 71 159 135 36 6 3 0 135 0 323 

Total 381 580 425 91 43 17 2 149 23 1005 

1 Publications appearing in print in FY 2004. Another 165 manuscripts have been submitted for publication. 2

Committee totals include 61 editorships and the TRC 3

CRADA’s signed this year only 

4

There are a total of 44 active patents. 

5

SRMs/RMs (Certificates issued) 

6

Calibrations were performed for over 306 customers. 

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

Lastly, economic impact studies, conducted by outside experts, investigate the impact of CSTL products and services on 

the US 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 US 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. CSTL has completed six of these 

studies between 1997 and 2003 to help evaluate the impact of its standards and data products. In collaboration with the 

Mayo Clinic, the impact of measurement uncertainty on PSA measurement was initiated in FY 2004. The first phase of 

this work, Standards for Calcium Testing, was completed last year and is described 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. 

 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 Cals. – Calibrations 
9

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 and 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, published in 2003, 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 US, 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. 

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 highquality 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 US 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. NIST has 

proposed to analyze individual NTRM gas cylinders and provide individual uncertainty analyses 

for each cylinder of the standard requested; these 

products would be known as NTRM Prime. This is 

driven by market demand for a “better product”; 

improvement in bottom-line profit by avoiding 

redundant analyses; and the desire to anticipate 

future changes in regulatory requirements particularly as they relate to emissions trading. 

This is the first of a three-part 

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. 
10

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 US more 

than one trillion dollars are spent each year on health care, which is about 14 % of 

the US gross domestic product (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. 

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 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 US-based companies 

supply 60 % of the IVD products sold in Europe, it is critical to US 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. NIST led the efforts in an international working group on 

Reference Materials and Reference Laboratory Procedures in 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 EU IVD Directive regarding in vitro diagnostic medical 

devices. Details are provided in the Measurement Standards section of this report. 

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. 

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. 
11

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 dieselfueled 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, SRM 1616b Kerosene, to 

be released soon, has certified sulfur levels at approximately 8 

μg/g, the lowest level of any Certified Reference Material 

available worldwide. 

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 and the resulting requirement 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 equipment, key in applying 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. Version 7.0 is the current 

public release, and version 8.0 is in beta testing with release expected in 

mid-2005. 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. Version 8.0 

A testimony on the relevance of NIST low-sulfur 

in Diesel Fuel SRMs 

“The development of (diesel fuel) SRMs directly supports the introduction of ultra-lowlevel diesel fuel with the implementation of the 

US EPA2007 highway heavy-duty and 2010 

Tier 4 non-road diesel regulations. These regulations when fully implemented will provide 

roughly $150 billion annually in health and 

welfare benefits to the American Public.” 

Margo Tsirigotis Oge, Director 

Office of Transportation and Air Quality, EPA

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

pointed their customers to 

REFPROP and CYCLE-D, and 

REFPROP has been licensed for use 

with the ASPEN and FLUENT 

process simulation package. 
12

will greatly expand the list of fluids, including cryogenic fluids, 

hydrocarbons, and simple inorganics in addition to refrigerants. 

This expansion was already anticipated by the new title beginning with version 7.0: Reference Fluid Thermodynamic and 

Transport Properties with “Reference Fluid” replacing the “Refrigerant” of earlier versions. 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. ISO recognizes the high 

quality of REFPROP and therefore specifies the same models 

(and thus the same property values) in its recently approved 

standard on refrigerant properties. 

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 US thermocouple industry (suppliers of wire 

and thermocouple assemblies) into the US 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. 

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 US manufacturers. 

Now available online at

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

CSTL researchers will port the computational engine of REFPROP to the ThermoData Engine (TDE), a new NIST database 

providing on-demand correlation of experimental data. This will give TDE the 

advantage of the inherent thermodynamic 

consistency of representing properties 

with equations-of -state versus the present 

approach of representing different properties with discrete correlations. Models for 

aqueous (water-based) systems and hydrocarbon mixtures containing hydrogen and 

helium will also be developed. 
13

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 FY2004. 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/ 
14
15

B. CSTL Programs 
16
 17 

1. Automotive and Aerospace (Transportation) 

According to the Bureau of Transportation Statistics, in 2002 Americans traveled 

over 5 billion passenger miles (i.e., 18,000 miles/person). From those, 90% were 

traveled in our highway system, while 9% was traveled in our airways. 

Recognizing the importance of the transportation industry to our national economy, CSTL provides standards for emissions testing and flow measurements to 

help the automotive and aerospace sectors meet their 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 hightechnology jet engines. 

Gas Mixtures Standards for the Automotive Industry: The NTRM Prime Program 

W.D. Dorko (839) 

Increasingly stringent environmental regulations place continuous demands on 

automobile manufacturers to improve the performance and quality of their vehicles. Among the requirements of these regulations is the traceability of emission 

test results to national standards. Through the NIST Standard Reference Material 

(SRM) and NIST Traceable Reference Material (NTRM) programs, CSTL helps 

support the traceability of gas standards needed by the automobile industry and 

other sectors of the US economy. 

As part of the NTRM program, Specialty Gas Companies produce – in concurrence with NIST – batches of gas mixtures. The composition of these mixtures is 

analyzed and results are sent to NIST for review. NIST also analyzes 10% of the 

cylinders produced to ensure the quality of the claimed results. The concentrations of the components in the gas mixture are assigned based on NIST results 

and their uncertainties are estimated using both the NIST and Specialty Gas Producer data. 

Recently, the automotive industry requested that NIST 

provide gas mixtures with uncertainties lower than those 

claimed for the NTRM products. One response would be 

for NIST to analyze individual NTRM gas cylinders and provide individual uncertainty analyses for each cylinder of the standard requested; these products 

would be known as NTRM Prime. A meeting of all interested parties will be held 

at the 2005 Pittsburgh Conference to further refine the scope of the proposed 

NTRM Prime program. 

In the future, the automobile industry and other larger users of NTRMs will decide which gas mixtures are needed. NTRM producers are expected to manufacture the required products per industrial requests. 

When the California Low Emissions 

Vehicle (LEVII) 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. 

NIST will support NTRM 

manufacturers in providing the products necessary 

to meet regulatory requirements by increasing 

the number and concentration ranges of gas mixtures 

for which it maintains primary standards. 
 18 

New Hydrocarbon Liquid Flow Standard for 0.2 L/min to 5 L/min 

T. T. Yeh, P. I. Espina, and J. Aguilera (836) 

Turbine-based power plants are heavily used in ships, aircraft, large 

ground vehicles, and portable electric plants by the US Department 

of Defense (DoD) and the commercial aircraft industry. Critical requirements for these include the performance of liquid flow meters 

used in turbine engine test stands. 

The testing of modern, high-efficiency engines requires uncertainties 

that have begun to exceed the capabilities of NIST’s liquid hydrocarbon flow standards which have an uncertainty of 0.12 % of reading. This prompted the DoD Calibration Coordination Group (CCG) 

to award NIST a grant aimed at developing a new generation of 

standards capable of providing traceability with uncertainties no 

larger than 0.025 % of reading. Recently we successfully concluded 

the first phase of this program, and today NIST has a new flow calibrator (see figure) designed to provide the requested performance. 

NIST procured a volumetric displacement flow standard (also known 

as a piston prover) from Flow Dynamics Inc. (Phoenix, Arizona) – 

currently the only manufacturer of such systems in the US. Design 

features and specifications for the new flow standard were collaboratively selected by staff from NIST and the commercial supplier. 

Upon receipt of the standard, NIST completed an extensive effort to 

establish direct traceability for the time, temperature, and length 

measurements that are used by the standard to assess the liquid flow. 

A detailed uncertainty analysis for the flow standard was written and 

tests were conducted to validate its conclusions. Using this system, 

the NIST measurement uncertainty for hydrocarbon liquid flows has 

been reduced by more than a factor of ten to 0.01 % (k=2). 

The new 2-liter-capacity hydrocarbon liquid flow standard replaces a 

gravimetric system over the range of 0.2 L/min to 5 L/min. It evaluates 

the performance of flow meters using type II hydrocarbon calibration 

fluid that simulates flow meter characteristics in jet fuel by closely 

matching fluid viscosity and density values. The system is fully automated; once the operator specifies flow set points, the data are accumulated and stored for later analysis and report generation. This automation 

reduces data transcription errors and calibration costs for our customers. 

In the coming year we will procure a 20 L Hydrocarbon Liquid Flow Standard and characterize it to cover flows from 2 L/min to 50 L/min. 

Ultimately, the new systems will replace our 

outdated gravimetric primary standard which 

has served this calibration service since 1954. 

CSTL researchers improve the 

uncertainty of NIST’s hydrocarbon liquid flow standards by 

more than a factor of 10. 

A dual rotor turbine meter is installed in 

the test section of the 2 L Hydrocarbon 

Liquid Flow Standard. 

T.T. Yeh, P.I. Espina, G.E. Mattingly, N.R. Briggs, and J. 

Aguilera, “An Uncertainty Analysis of a NIST Hydrocarbon 

Liquid Flow Calibration Facility”, Proceedings of the 2004 

Heat Transfer / Fluids Engineering Summer Conf. July 2004.

NIST hydrocarbon flow measurement standards support the needs 

of aircraft turbine engine manufacturing requirements for improved fuel efficiency, supporting 

military and commercial user requirements 
 19 

Propulsion Systems Demand Accurate Property Data 

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

(838), I.M. Abdulagatov (Russian Academy of Sciences), and P.C. Andersen (2B Technologies) 

As part of the Next Generation Launch Technology Program, 

NASA is designing advanced rocket engines that will combust a 

kerosene-based fuel known as RP-1. RP-1 is a complex liquid 

fuel that consists of more than 100 components, for which present-day thermophysical properties models are based on very 

limited data. NASA has concluded that RP-1 property estimation accounts for 70% of the uncertainty in the propulsion system design. NIST used a gas chromatography–mass spectrometry–infrared spectrophotometry method to chemically 

characterize RP-1. From its constituents, 20 compounds were selected as part of a surrogate mixture used for measuring 

and modeling thermophysical properties. Measurements of chemical composition, boiling temperature, density, heat 

capacity, viscosity, and thermal conductivity were conducted in a range of temperatures up to 700 K and pressures to 60 

MPa. 

Short equations of state and models for the transport 

properties were developed and implemented in a 

user-friendly program. 

These accomplishments were presented to rocket 

fuels specialists during a workshop hosted at the 

NIST Boulder campus in December 2003. Participants from NASA, the Air Force, commercial 

rocket engine manufacturers, and academia were 

eager to use the new results, and interested in continued NIST efforts to explore other features related 

to fuel properties. 

New Gas Standards for Calibrating Instrumentation Used for Measuring Emissions from 

Next-Generation Low-Emission Vehicles 

W.J. Thorn III (839) 

Since 1975, NIST and the US Motor Vehicles Manufacturers have worked together to develop gaseous Standard Reference Materials (SRMs), which by federal law are the Nation’s standards for all mandated fuel economy and mobilesource emission measurements. Currently, the American Industry/Government Emissions Research consortium 

(AIGER) is working to help the automobile industry meet 2003 Federal Tier II and California LEV II emission regulations. NIST supports AIGER by maintaining inventories of required gas SRMs that consist of 

dilute mixtures of key pollutants such as hydrocarbons, carbon monoxide, and nitric oxide. 

0.070

0.080

0.090

0.100

0.110

0.120

0.130

0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85

ρ / g cm-3

λ / W m -1K -1

300 K

350 K

400 K

450 K

500 K

547 K

608 K

650 K

Accurate experimental results for thermal conductivity 

of RP-1 have replaced estimated values NASA had relied 

on since the 1950s

NIST improves the accuracy of thermophysical properties of RFP-1 by developing new measurements and models, removing a key source of uncertainty in 

propulsion system design. 
 20 

In 1998, NIST worked with a Specialty Gas contractor to blend cylinders of low NO standards at concentrations ranging 

from 0.5 μmol/mol to 1.25 μmol/mol. The new standards exhibited excellent stability for more than 5 years and final 

concentration values with associated uncertainties were recently assigned. In 2001, AIGER requested the production of 

two new low NO SRMs at concentrations of 0.5 μmol/mol and 1.0 μmol/mol (SRMs 2737 and 2738 respectively). 

NIST and a contractor prepare the new working standards using NO in 

nitrogen blends. The NIST certification analysis for the new SRMs was 

completed in September 2004. 

Driven by future requirements by AIGER, NIST continues to move NO 

analytical standards and measurement capability below 0.1 μmol/mol levels. 

Combustion Simulation Databases for Real Transportation Fuels: A New Community Collaboration 

T. C. Allison, D. R. Burgess, Jr., J. W. Hudgens, W. Tsang, and J. A. Manion (838) 

The economic, environmental, and health benefits that could be derived from improved combustion processes are enormous and well 

recognized. In September 2003, the leading experts from industry, 

academia, and government met at the NIST Workshop on Combustion Simulation Databases for Real Transportation Fuels to assess 

needs and opportunities to translate scientific understanding of combustion to technological development. Attendees affirmed the value 

of predictive models of combustion in the development of less polluting engines. The workshop concluded that a coherent effort is 

required to address the complexities associated with the combustion 

of real transportation fuels and that a standard infrastructure for the 

exchange of combustion-related data must be developed. 

As a result of the workshop, NIST joined with other members of the 

combustion community in the PrIMe (Process Informatics Model) 

collaboration. The goal of PrIMe is to generate standard formats for 

combustion data, together with data libraries, analysis software tools, 

and the supporting IT infrastructure that will enable development of 

predictive models for combustion. The PrIMe uses the DoE supported (CMCS) project to develop and support the IT infrastructure 

required for the effort. NIST’s commitment to PrIMe envisions the 

creation of new paradigms for scientific collaboration involving the 

construction of a traceable, comprehensive electronic repository of 

key data relevant to modeling combustion processes. 

The initial data library consists of data from the NIST Chemical Kinetics Database and GRI-Mech 3.0 (the current 

“standard” model for combustion of small hydrocarbons). The library includes data collections on atomic properties, 

chemical species, thermodynamics, elementary kinetics, and transport, as well as bibliographic data. A collaborative 

workspace for the PrIMe project has been created within the CMCS portal. 

Launching of the initial PrIMe data repository is anticipated in the 

first half of 2005. Longer-term plans include the integration of 

sophisticated tools that reduce human efforts in model creation and 

reduction. 

NIST releases two new low-level 

nitrous oxide SRMs 

SRM 2737 − 0.5 μmol/mol 

SRM 2738 − 1.0 μmol/mol 

Ultimately the PrIMe project will facilitate the rapid construction of predictive 

models for combustion chemistry. 

http://kinetics.nist.gov/RealFuels/

NIST/CSTL leads the creation of a 

standard infrastructure for the exchange of combustion data, which is 

critical to technological development. 
21

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. 

Biocompatible materials used in the worldwide $50 billion end-use device 

market account for current annual sales of approximately $1 billion. Enduse devices include implants, valves, grafts, pacemakers, bone repair and 

replacement devices, artificial organs, dental materials, drug-delivery systems, dialysis/separation/filtration systems, and catheters and stents. 

 

Functional Reconstitution of Ion Channels in Polymerizable Lipid Membranes 

D. K. Shenoy, W. Barger, A. Singh (NRL), R. G. Panchal (USAMRIID), M. Misakian (EEEL), V. M. Stanford 

(ITL), and J. J. Kasianowicz (831)

For the past four decades, the art of making planar lipid bilayer membranes has 

enabled the study of ion and macromolecular transport through single or multiple 

nanometer-scale pores. It is in such matrices that nanopore ion channels, which are 

the molecular basis of many cellular functions (e.g., nerve and muscle activity), 

have been shown to be capable of detecting and characterizing different ions, 

polynucleotides, and particular proteins. Because only weak intermolecular interactions stabilize liquid-crystalline phospholipid membranes (i.e., the membranes 

are too fragile), nanopore-based applications would benefit if ion channels could be functionally reconstituted and immobilized in robust ultra-thin films. Toward that goal, this study demonstrates that two different protein ion channels 

(formed by Bacillus anthracis protective antigen 63 and Staphylococcus aureus α-hemolysin) are fully functional in two 

different polymerizable lipid membranes in the liquid crystalline state. In addition, one of these channels functions even 

after the membrane is partially polymerized. Both the anthrax channel and the α-hemolysin channel spontaneously 

formed highly conducting nanopores in membranes formed by either of 

two polymerizable lipids (the top two structures shown to the right; 

DC8,9PC − 1,2-bis(10,12-tricosadiynoyl)sn-Glycero-3-Phosphocholine, 

and PTPE − 1-Palmitoyl-2-10,12 Tricosadiynoyl-sn-Glycero-3-Phosphoethanolamine) and the nonpolymerizable control phospholipid diphytanoyl phosphatidylcholine (DiPhyPC). Exposure of the polymerizable 

lipids to UV light causes diynes in the hydrocarbon chains to covalently 

link with others in like neighboring lipid molecules. 

The α-hemolysin ion channel remained completely functional in a PTPE 

bilayer membrane before and after the matrix was polymerized. The recordings in panel A show that in the absence of single-stranded DNA, the 

ionic current through a single α-hemolysin channel is quiescent. Adding 50-

nucleotide long poly(thymidine) causes transient current blockades that occur at random intervals. Following UV illumination of the PTPE membrane, 

the polynucleotide-induced current blockades persist. Five of the characteristic poly[dT]-induced blockades that occurred after UV illumination are 

shown in panel B. Note that there are three predominant states (fully open, α

and β). These states predominate before and after UV irradiation as is 

shown in the time series in panel C. 

Since recent work suggests the usefulness of the anthrax pore for rapid 

screening of anthrax therapeutics, the ability to functionally reconstitute 

such channels into matrices that can be made robust is promising. 

In the US, the number of people 

above 65 years is expected to 

more than double from 35 million 

in 2000 to 71 million in 2020, and 

the demand for biomaterials for 

the regeneration and repair of 

tissues and organs is expected to 

increase accordingly. 

Recent research conducted 

at NIST suggests that an anthrax pore might be useful 

for the rapid screening of 

anthrax therapeutics. 
22

Cluster SIMS Depth Profiling in Polymeric Blends for Protein Drug Delivery Applications 

C. Mahoney (837), P. Yu, and J. A. Gardella Jr. (State University of New York, Buffalo) 

Poly(L-lactic acid) (PLLA) has shown particular promise as a biodegradable material because the degradation product, lactic acid, is readily metabolised by the body. In addition, the degradation rate can be 

easily controlled through variation of its 

relative mass. Polyethylene oxide (PEO)-

containing copolymers, such as BASF’s 

Pluronic® surfactants (containing polypropylene oxide (PPO) and PEO components) are also useful polymeric materials for biomaterial and pharmacological applications as they are neutral, highly biocompatible and 

pharmacologically inactive water-soluble polymers. The incorporation of PEO-containing copolymers into biodegradable PLLA-based drug delivery implant systems is expected to improve the interfacial biocompatibility of the polymeric 

devices as a result of the preferential migration of the PEO component to the surface. In addition, blend matrices of 

PEO and relatively hydrophobic PLLA is also expected to improve the three dimensional stability and the biological 

activity of water-soluble macromolecular drugs such as proteins or enzymes in the delivery systems via micelle formation. When used as drug-releasing matrices, these PLLA/Pluronic® blends have been proven to extend protein release 

and minimize the initial protein burst when compared to the pure PLLA homopolymers. The composition in the subsurface region (10 nm -1000 nm) of these materials is highly important as it will determine the extent of initial burst 

release of any drugs present as well as the biocompatibility of the material. Until now, no methodology has been capable of yielding in-depth information from this region. 

The surface chemistry as determined by both X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion 

Mass spectrometry (TOF-SIMS) of these PLLA/Pluronic® blend materials indicates that there is an enrichment of the 

Pluronic® at the surface. However, these initial surface studies utilized monatomic primary ion beams, which cause 

significant subsurface damage particularly in organic and polymer samples. This increased beam-induced damage prevents the ability to obtain information as a function of depth in organic and polymer samples. Compared to conventional SIMS, “cluster SIMS” employing molecular rather than atomic primary ion beams often yields enhanced sensitivities, decreased accumulation of beam-induced damage, and increased sputter rates. These advantages 

have allowed us to obtain in-depth information from 

certain organic and polymeric materials for the first 

time. 

The figure shows the resulting in-depth profiles obtained from a 

PLLA/Pluronic blend system containing a mass fraction of 25% 

Pluronic®. These profiles are consistent with a surface enrichment 

of Pluronic®-P104 surfactant, followed by a depletion zone, and 

then finally a constant bulk composition region. This effect was 

consistent over a range of concentrations (1% to 25%). Normalized signal intensities associated with Pluronic® (m/z = 59), PLLA 

(m/z = 128), and Si (m/z = 28) are plotted as a function of increasing SF5

+

 primary ion dose 

Secondary Ion Mass Spectrometry 

(SIMS) methods developed at NIST 

provide in-depth information from 

polymeric blends and potentially relate the sub-surface composition to 

the performance characteristics in 

real devices. 

0.0 4.0x1014

8.0x10141.2x10151.6x10152.0x1015

0.01

0.1

1

10

100

Pluroni

PLLA 

Si

0.0 4.0x1014

8.0x10141.2x10151.6x10152.0x1015

0.01

0.1

1

10

100

Pluroni

PLLA 

Si

Normalized Intensity (log scale) 

vs Ion Dose (Ions/cm2

) 

The depth profile shown here represents the team’s first 

successful attempt to obtain in-depth information from 

polymeric blend systems using SIMS. 
23

Because of the well-behaved nature of these materials under cluster ion bombardment, we have also successfully obtained quantitative depth profiles. These results demonstrate that with cluster 

primary ion bombardment, we are now able to successfully monitor and quantify the preferential segregation that occurs within 

certain multi-component polymer blends. 

Future plans include the determination of the 3-D molecular structure of these and other drug delivery systems (such as 

drug eluting stents and insulin delivery systems). We plan to monitor the diffusion and release of proteins and drugs 

from these systems using cluster SIMS technology. In addition to this, collaborations are being established with pharmaceutical and biomedical device manufacturing industries in order to relate the 3-D compositional structure to the performance characteristics in real devices.

The development of cluster secondary ion mass 

spectrometry (SIMS) for in-depth analysis of polymeric biomaterials has potential long-term impact 

for quality control and product development in the 

biomedical/pharmaceutical arenas. 

0.0 3.0x1014

6.0x10149.0x1014 0

10

20

30

40

50

60

70

80

90

0.0 3.0x1014

6.0x10149.0x1014 0

10

20

30

40

50

60

70

80

90

Composition depth profile of Pluronic in 

PLLA using SIMS calibration. 

Normalized Intensity (log scale) 

vs Ion Dose (Ions/cm2

) 
24
25

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. 

A Proton-Coupled Dynamic Conformational Switch in the HIV-1 Dimerization Initiation Site Kissing 

Complex 

M.-R. Mihailescu (CARB/UMBI) and J. P. Marino (831) 

RNA and RNA-protein (RNP) complexes represent attractive targets for new drug therapies aimed at treating 

retroviral and bacterial infection. Modulation of RNAprotein interactions involved in retroviral gene expression 

could provide novel ways to combat viral infection, or 

enhance the effectiveness of existing antiviral agents. 

In the human immunodeficiency virus type 1 (HIV-1), the 

dimerization initiation site (DIS) is the sequence primarily 

responsible for initiating the non-covalent linkage of two homologous strands of genomic RNA during viral assembly. In 

a structural rearrangement catalyzed by the HIV-1 nucleocapsid protein (NCp7) and suggested to be associated with maturation of the budded viral particle, the DIS converts from a 

metastable kissing dimer to an extended duplex. Using fluorescence and NMR methods, we have demonstrate that the 

DIS kissing dimer displays localized conformational dynamics that result from the specific protonation of the N1 base 

nitrogen of the DIS loop residue A272 at near physiological 

pH. The rate of NCp7 catalyzed maturation of the DIS kissing dimer has also been shown to directly correlate with the 

observed proton-coupled conformational dynamics, where 

NCp7 is found to convert the dynamic A272 protonated state 

with a faster rate. Taken together, these results reveal a novel 

role for base protonation in modulating local RNA structure 

and demonstrate a mechanism for promoting the chaperone 

mediated structural rearrangement of a kinetically trapped 

RNA conformational state. 

The RNA conformational ‘switch’ identified in the HIV-1 DIS dimer 

represents a possible new target for antiviral drugs. Through collaboration with researchers at the HIV-1 Drug Resistance Program at the National Cancer Institute, researchers at CARB plan to screen large public 

domain libraries of low relative molecular mass compounds that target 

DIS and inhibit its functional role is genome dimerization and maturation. 

According to a recent Dept. of 

Commerce Report on the Pharmaceutical Industry, the US Government action has focused on creating 

the environment that would best 

encourage further innovation and 

yield a constant flow of new and 

innovative medicines to market. The 

goal has been to ensure that consumers would benefit from technological breakthroughs ... 

NIST research focuses on the development 

of general approaches for detecting and 

quantifying RNA-protein and RNA-small 

molecule interactions, which can be employed in high-throughput screens (HTS) 

and for obtaining rapid structural information to guide rational drug design. 

Plot of the normalized fluorescence decay as 

a function of time after NCp7 protein was 

added to a DIS kissing complex at pH 6.0 

(red circles) and 7.2 (blue squares). Inset is a 

schematic of the NCp7 catalyzed structural 

isomerization of the kissing dimer. Asterisks 

indicate the position of 2-AP in the 

DIS24(GA)-4ap stem-loop. 

Recent publication on this research:

M.R. Mihailescu and J. P. Marino 

(2004) Proc. Natl. Acad. Sci., USA., 

101(5), 1189-1194. 
26

Leveraging Traceability for Chemical Spectrophotometry Through the Commercial Sector

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

Recognizing that the NIST model has been successfully 

emulated in commercially-produced traceable CRMs, 

NIST is terminating the production of individuallycertified neutral-density filter Standard Reference Materials® (SRMs®) for chemical spectrophotometry. Sales 

of SRMs 930e, 1930, and 2930 (0.001 < T < 0.9, neutral-density glass filters for the visible spectrum) will 

cease when existing stocks are depleted. Production of 

SRM 2031a (0.1 < T < 0.9, metal-on-fused-silica neutral-density filters for the UV and visible spectral regions) will be curtailed to end sales on schedule with 

the glass filters. 

End users are encouraged to purchase CRMs from secondary suppliers who are 

appropriately accredited to Guide 17025 or Guide 34 of the ISO, or who possess 

documentation consistent with the NIST traceability policy. Such commercial 

suppliers may also recertify expired NIST SRMs, although NIST will continue to 

offer recertification for the 3500+ sets of SRM filters in the field. 

In a related move, transmittance and absorbance measurements, used in solid filter 

recertification and future batch SRM production, will soon feature traceability 

through the regular transmittance scale maintained by NIST. This will result in 

a single U.S. national scale for regular spectral transmittance measurements 

conducted in support of both physical and chemical metrology. This 

transmittance scale is routinely compared to other 

national transmittance scales through measurements 

coordinated by the Comité Consultatif de Photométrie et 

Radiométrie (CCPR) of the Bureau International des Poids 

et Mesures (BIPM). 

In the long term, these changes are expected to benefit affected industries (e.g. pharmaceuticals, chemical products, and health technologies) 

by supporting a robust and responsive system of fit-for-purpose reference materials that are manufactured by competing commercial 

sources. NIST is attempting to avoid near-term anxiety in the heavily 

regulated pharmaceutical sector by implementing extensive educational 

activities during the one- to two-year period required to sell out the 

stocks of these filters. 

Thermodynamics of the Hydrolysis Reactions of Nitriles 

Y. B. Tewari and R. N. Goldberg (831) 

The use of nitrilase enzymes has attracted substantial interest in the 

biotechnology community because conventional chemical methods 

for nitrile hydrolysis entail the use of harsh conditions such as the 

use of concentrated acids or bases and high temperatures. Such 

harsh conditions are generally not useful when sensitive complex 

molecules or chiral compounds are involved. Most importantly, 

the chemical methods do not permit the asymmetric synthesis of 

chiral compounds. In contrast, the nitrilase-catalyzed reactions 

proceed under mild conditions and produce a high yield of a 

stereospecific product. The importance of nitrilase enzymes has 

led recently to the commercial development of a library consisting 

of over 200 new forms of this enzyme. 

NIST provides thermodynamic data 

for nitrilase enzyme reactions. The nitrilase enzymes catalyze the direct hydrolysis of organic nitriles to the corresponding carboxylic acids, which are 

of increasing interest to the pharmaceutical, and other multi-billion dollar 

industries. 

The controlled termination of NIST’s longstanding program for the individual certification 

of spectrophotometric absorbance standards will 

capitalize on the International Organization for 

Standardization (ISO)/NIST description of traceability in order to stimulate a more efficient and 

leveraged model for supplying certified reference 

materials (CRMs) for absorbance. 

In order to facilitate the transition, 

NIST has widely publicize these changes 

by making presentations at FACSS 2004 

and EAS 2004 as well as providing information on the NIST web site and in 

anticipated announcements in trade 

magazines. 
27

Several representative (model) nitrilase-catalyzed reactions were selected 

for this investigation, which used HPLC and calorimetry combined with 

equilibrium modeling calculations. In all cases the reactions proceeded 

to completion. Specifically, the HPLC results (forward and reverse reactions) gave values of the apparent equilibrium constant K’ having a lower 

limit of 350. A thermochemical cycle calculation showed that a value of 

the equilibrium constant for a typical ionic reference reaction for this 

class of reactions is on the order of 1011. Values of the standard molar 

enthalpy of reaction ∆rHm° for the ionic reference reactions ranged from -77 kJ·mol-1 to -99 kJ·mol-1. The thermodynamic results obtained in this study provide quantitative data that can be used for the bioprocess engineering (design 

of bioreactors and conditions of reaction) of these enzyme-catalyzed reactions. The results are the first to be reported 

in the literature. 

2 H2O NH3

H

COOH

CH3

H

C

CH3

N

(R) (R)

α-Methylbenzylcyanide α-Methylbenzeneacetic acid

+ +

Chorismate Pathway Enzymes: Structural Studies 

J.E. Ladner (831), E. Eisenstein, J. Parsons, and K. Calabrese (CARB/UMBI) 

Aromatic hydrocarbons are difficult to produce synthetically, however the 

elucidation of natural biochemical pathways makes it easier to alter and 

utilize these pathways to make these and similar chemicals. In particular, 

the phenazines, which are produced by a branch of the chorismate pathway in some bacteria, are difficult to synthesize but are important potential drugs. Precise enzymatic mechanisms, and relationships between the 

structure and the physical properties can be predicted only when the detailed three-dimensional structures are known for the enzymes. 

Several species of Pseudomonas, including the human pathogen P. aeruginosa, produce secondary metabolites known 

as phenazines. Dozens of naturally occurring phenazines have been described, all of which share the characteristic 

tricyclic heteroaromatic ring system. Phenazines are redox active compounds that participate in reactions yielding 

superoxide and peroxide ions, and hydroxyl radicals. These toxic molecules are thought to control the growth of other 

microorganisms, provide Pseudomonas with a competitive growth advantage, and may enhance the ability of these 

pathogens to colonize human and other tissue. 

Two operons in Pseudomonas aeruginosa, each containing seven genes, are involved in the biosynthesis of phenazine 

(phzA-G). A similar, single operon has been described in P. fluorescens 2-79. Each of these operons encode all of the 

genes required to produce phenazine-1-carboxylic acid (PCA) from chorismate. This research provides a better understanding of the phenazine pathway. Structures 

of the products of genes phzF, phzG, and 

phzD have been solved, and both biochemical 

and crystallographic methods to determine 

their roles, substrates, and products have been 

pursued. As the research moves forward 

enzymes that are homologues of these 

enzymes from other related pathways will be 

studied, and work to try to understand the 

details of how the enzymes perform their 

specialized tasks will continue. 

The nitrilase-catalyzed hydrolysis of 

α-methylbenzylcyanide to αmethylbenzeneacetic acid and ammonia 

Publication in Press: 

Y.B. Tewari and R.N. Goldberg, 

“Thermodynamics of the hydrolysis 

reactions of nitriles”, The Journal of 

Chemical Thermodynamics. 

NIST studies how aromatic hydrocarbons are produced enzymatically in bacteria. This can be 

of great benefit to the pharmaceutical industry, and ultimately 

the public health. 

Recent Technical Publications: 

1. J.F. Parsons, F. Song, L. Parsons, K. Calabrese, E. 

Eisenstein, J.E Ladner, “Structure and Function of the 

Phenazine Biosynthesis Protein PhzF from Pseudomonas fluorescens” 2-79. Biochemistry 43, (2004). 

2. J.F. Parsons, K. Calabrese, E. Eisenstein, J.E. Ladner, 

“Structure of the Phenazine Biosynthesis Enzyme PhzG”

Acta Crystallographica, D60, (2004) 
28
29

4. Chemical and Allied Products 

NIST/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, world-recognized 

standards. 

For the past several years, high energy costs 

had siphoned profits for the chemical industry 

despite the increase in demand for products. 

However 2003 showed improvements in profits and 2004 began with major US chemical 

companies posting their best results in years. According to a C&E News Report in December 2004, the “upturn is no longer elusive, and although challenges remain, prosperity is 

finally returning to the chemical industry”. 

IUPAC Partnership Develops Standards and a Data Retrieval System for Ionic Liquids 

J.W. Magee, J.A. Widegren, D.G. Archer, S.L. Outcalt, M.Frenkel, R.D. Chirico, Q. Dong, A. Laesecke, 

M.O. McLinden, R.A. Perkins (838), K.N. Marsh (U. of Canterbury, New Zealand), B.-C. Lee (Hannam U., Korea), and E.M. Saurer (U. of Notre Dame) 

Ionic liquids, a class of organic salts that are liquid at or near room temperature, have been proposed as solvents for Green Processing. In spite of the 

many advantages that these fluids are predicted to offer, fundamental data 

on their physical and chemical properties, property measurement methodology, high quality data on reference systems, standards for reporting thermodynamic data, and creation of a comprehensive database infrastructure 

for ionic liquids are needed to provide US industry with a knowledge base 

to exploit these solvents. 

To achieve these goals in the shortest time possible and reach an international consensus on the central issues, two IUPAC projects have been initiated with 

NIST/CSTL involvement. The first of these IUPAC projects is Thermodynamics 

of Ionic Liquids, Ionic Liquid Mixtures, and the Development of Standardized Systems. The lack of consistent requirements for the publication of thermodynamic 

data for ionic liquids has led 

to major barriers to an unambiguous interpretation of 

the data and a critical 

evaluation with regard to their uncertainties. This drastically diminishes the value of the reported numerical data for use in a variety of engineering applications. To address these issues, a standardization in reporting thermodynamic data for ionic liquids 

could be accomplished by an expansion of the Guided Data Capture (GDC) software developed by the NIST’s TRC Group, formerly known as the Thermodynamic Research Center (TRC). 

Outcomes of the first workshop were reported in a special section of the Journal of Chemical and Engineering Data. In 

Beijing, the IUPAC Task Group planned an international 

round robin study of a reference substance, 1-hexyl-3-

methylimidazolium bis(trifluoromethylsulfonyl)amide, abbreviated as [hmim][Tf2N]. Using NIST-supplied reagents, 

1.5 kg of [hmim][Tf2N] was synthesized at the University of 

Notre Dame and then was shipped to NIST. 

Often referred to as the keystone 

industry because nearly every 

sector of the manufacturing economy uses its products, the US 

chemical industry ranks among 

the world’s leading industries, 

accounting for an estimated onequarter of total world chemical 

production. 

This IUPAC task group has convened international discussion of the issues by conducting two IUPAC workshops on ionic liquids, 

held at the 17th IUPAC Conference on 

Chemical Thermodynamics (Rostock, Germany) at the 18th IUPAC Conference on 

Chemical Thermodynamics (Beijing, China) 

and a 3rd Workshop will be convened in 

Boulder as part of the 19th IUPAC Conference on Chemical Thermodynamics in 2006. 

NIST works with the international community to address the 

lack of consistent requirements 

for reporting thermodynamic 

data for ionic liquids by employing the Guided Data Capture 

software developed in CSTL. 

International Union of Pure 

and Applied Chemistry 

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. 
30

After drying and chemical characterization at NIST, samples were packaged under an inert atmosphere and were 

shipped to the participating laboratories. NIST will coordinate round robin studies of density, heat capacity, and viscosity, and will participate in measurements of those properties plus thermal conductivity, electrolytic conductivity and 

industrial gas solubility. 

The second IUPAC project Ionic Liquids Database is addressing the need for an open-access, public-domain data storage system scoped to cover information pertaining to ionic liquids. The vision for this project is to create a distributedaccess data retrieval system for ionic liquids and their mixtures that encompasses chemical structure, solvent properties, 

ionic liquids use in synthesis, reviews, reactions and catalysis, manufacturer information, benchmark properties and 

models, and thermophysical and thermochemical data. During FY 2004, the task group met to share a common vision 

and to divide the data collection effort among the participants. 

The NIST measurement program continues to 

provide benchmark physical properties data 

for selected ionic liquids that are liquid-phase 

at room temperature and both air- and moisture- stable. For 1-butyl-3-methylimidazolium 

hexafluorophosphate, considered to be the 

archetypal ionic liquid, we have published the 

first reports of thermodynamic properties of 

the ideal gas 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 K to 550 K. A study of the viscosity of three hydrophobic ionic liquids and the effect of a dilute water impurity has been completed. 

In the future, properties studies will continue with new 

measurements of thermodynamic density, heat capacity, 

enthalpy of solution, gas solubility, and also expanded 

transport property measurements, such as thermal conductivity and electrolytic conductivity, a key electrical characteristic. Structure-property relationships for physical properties will be explored by analyzing evaluated data in our 

database. Modifications that support ionic materials will 

be applied to the TRC Source Database at NIST and to the 

Guided Data Capture application to facilitate the storage 

and retrieval of ionic liquids property data. 

Second Industrial Fluid Properties Simulation Challenge 

R.D. Mountain, D. Friend, R.D. Johnson, A.M. Chaka, F.Case (Case Scientific), D. Frurip, J. Moore, J. Olson 

(Dow), J. Golab, (BP Amoco), P. Gordon (ExxonMobil), P. Kolar (Mitsubishi Chemical), R. Ross (3M), and 

M. Schiller (DuPont) 

The Industrial Fluid Properties Simulation Challenge is an opportunity for the molecular simulation community to predict fluid 

properties that are not available in the open literature. This competition is an open, biannual program that is sponsored by the Computational Molecular Science and Engineering Forum (CoMSEF) 

of the American Institute of Chemical Engineers (AIChE) and by 

the Theoretical Subdivision of the American Chemical Society 

(ACS) Physical Chemistry Division. 

Y.U. Paulechka, G.J. Kabo, A.V. Blokhin, O.A Vydrov, J.W. 

Magee, and M. Frenkel, “Thermodynamic Properties of 1-

Butyl-3-methylimidazolium Hexafluorophosphate in the Ideal 

Gas State” J. Chem. Eng. Data 2003, 48, 457-462. 

G. J. Kabo, A. V. Blohkin, Y.U. Paulechka, A.G. Kabo, M.P. 

Shymanovich, and J.W. Magee, “Thermodynamic Properties 

of 1-Butyl-3-methylimidazolium Hexafluorophosphate in the 

Condensed State” J. Chem. Eng. Data 2004, 49, 453-461. 

Anomalously high sensitivity of transport properties (in this case kinematic viscosity) to water 

content has been revealed by recent NIST measurements on ionic liquids. 

NIST in collaboration with scientists at 

Dow, BP Amoco, Case, Mitsubishi Chemical, 3M, and DuPont have organized the 

Second Industrial Fluid Properties Simulation Challenge. 

-40%

-35%

-30%

-25%

-20%

-15%

-10%

-5%

0%

5%

0.0% 0.3% 0.6% 0.9% 1.2% 1.5%

Water content of the ionic liquid 

Change in viscosity 

[C2mim][Tf2N] 

[C4mim][Tf2N] 

[C4mim][PF6]
31

The goal of the Second Industrial Fluid Properties Simulation Challenge was to evaluate and benchmark the available 

molecular simulation methods and force fields on problems that have significant industrial relevance. This process will 

encourage the continued development of better algorithms, methods, and force fields, while improving the alignment of 

academic efforts with industrial needs. Molecular simulation has been identified as a promising technology for predicting thermophysical properties in the Vision 2020 Roadmap for the Chemical Industry. The industrial participation 

within the organizing committee was even stronger than for the first challenge, and the financial contributions from this 

sector, which were applied largely for prizes for the most successful entrants, were substantial. Further, the number and 

quality of the entries submitted to the challenge indicated an increased interest in the process, and the contest has attained a truly international stature, with entrants, judges, and organizers from around the globe. 

The three problems in the challenge were announced at the 2003 Fall ACS meeting. Entrants had one calendar year to 

complete their projects. Benchmark values and uncertainties for the specific problems presented were 

determined by scientists at NIST and Dow Chemicals, through a combination of new experiments and 

extensive evaluation of available information. These 

benchmark results were used to evaluate the second 

challenge entries. There were five entries in problem 

1, five entries in problem 2, and two entries in problem 3. 

The entries were evaluated in a two-step, double blind process. In the first step, experts in the area of molecular simulation, who were not associated with the organizers and were not entrants, evaluated the overall scientific quality of the 

entries. Then the entries were ranked by the organizing committee in terms of how accurately the simulation based predictions of fluid properties reproduced the experimental values. The experimental values were not available in the open 

literature so the entries were predictions. 

The results of the competition were announced at 

a special session of the National Meeting of the 

AIChE in November 2004. For some of the 

problems, the entrants were able to produce reasonable predictions for the unknowns, at a level nearly adequate for some industrial purposes. However, for the most 

challenging problems, especially heats of mixing of the assigned aqueous system, considerable work will be needed 

before the technique will produce reliable results. 

Computational Chemistry Illuminates Atomistic Processes at Complex Interfaces 

A.M. Chaka (838) 

The chemistry of interfaces is of fundamental importance for a wide variety of environmental, industrial, and biological 

processes such as migration of pollutants in groundwater, catalysis, microelectronics, corrosion, and sensors. Progress 

in understanding these processes has been limited because the traditional techniques of surface science require ultrahigh vacuum 

(UHV) and clean, ideal crystalline surfaces, which may have little 

resemblance to real surfaces in complex environments. Recent 

advances in experimental surface science, however, have made it 

possible to conduct experiments on surfaces exposed to high concentrations of chemical species. These new instrument capabilities 

have revealed dramatic changes in surface chemistry upon exposure to high concentrations of reactants, as indicated by surface 

structural relaxations and Scanning Tunneling Microscope currents (which measure electronic energy levels), but are 

incapable of identifying the chemical structures or processes responsible. 

The situation in quantum theory for surfaces was similar to that in experimental, for example, electronic structure calculation by Density Functional Theory (DFT) is well established for calculating surface structure and energy only at 0 K 

under vacuum. We have collaborated in the development of new theoretical methods to extend DFT quantum mechanics 

to capture the thermodynamics and structure of real surfaces in equilibrium with multiple species in the environment at 

finite temperatures and pressures. 

Challenge 2003 was to predict: 

• vapor pressure and heat of vaporization (problem 1) 

• Henry's law constants (problem 2) 

• heats of mixing for specific materials and conditions 

(problem 3) 

More information is found on the website at 

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

NIST partners with key experimental 

groups to illuminate the chemistry occurring at complex metal oxide surfaces and to extend and validate the 

theoretical methods developed to predict and understand the surface chemistry for “real” systems. 
32

The hematite (0001) surface, α-Fe2O3, is important for many reasons. It is the catalytic surface used commercially to 

convert ethyl benzene to styrene, the most prevalent mineral surface exposed to ground and surface water, and is a 

model surface to study corrosion for ferrous-based metals. The first high-oxygen-pressure STM work on the hematite 

(0001) surface indicated the presence of two distinct chemical domains. These were first interpreted as distinct iron- and 

oxygen-terminated regions with the Fe-O3-Fe-R and O3-Fe-Fe-R structures, indicated by cleavage planes shown in the 

figure. 

More recent experimental work and our theoretical surface energy calculations indicated that the lowest energy surface 

that would be formed under the experimental conditions was not the O3-structure but was a O=Fe (ferryl) structure that 

resulted from dissociation of oxygen on the Fe-terminated surface. 

The predicted threshold pressure and temperature for the formation of Fe=O was 

in quantitative agreement with experiment, further validating the first-principles 

methods used in the model. Theoretical calculations also obtained a Fe=O vibrational stretching frequency of 981 cm-1 in excellent agreement with the 989 cm-1 

band observed by infrared reflection absorption spectroscopy (IRAS). The power of computational chemistry has provided great insight and discovery in surface chemistry that had not been previously observed on an iron oxide surface, 

and a manuscript describing this work has been submitted to Physical Review Letters. 

In another example of the value of our methodology for calculating surface chemistry, we addressed the reactivity of 

water at surfaces. Recently, scientists at Stanford University, University of Chicago, Lawrence Berkeley National Lab, 

and University of Alaska Fairbanks, made the puzzling observation that hematite is much more reactive towards trace 

amounts of water than the isostructural α-Al2O3 (corundum, sapphire), yet is more stable to weathering in nature. Washing the cleaved native hematite mineral surface with dilute acid and immersing it in water resulted in formation of two 

distinct (one dominant and one minor) domains of unknown chemical structure but with significantly different surface 

structure relaxations. 

α-Fe2O3 bulk unit cell and surface models 

for the three possible “clean” terminations 

that can result from cleavage. Large 

spheres are O and small spheres are Fe. 

The surface models are annotated with the 

labeling scheme used to identify the various atomic layers and the layer spacings 

for ideal (unrelaxed) atomic positions. 

High resolution STM image of the α-Fe2O3 (0001) film prepared at 

the FHI in 10 Pa (10-2 mbar) of O2 at 1020 K (size 30 x 30 nm2

, Vt = 

1.4 V, I = 1 nA) showing the multiple domains of iron- and oxygentermination. The lattice of protrusions with approximately 5 Å periodicity is clearly seen in the inset (size 3 nm2 x 3 nm2

). The unit cell 

is indicated. 
33

To identify these structures and understand the differences 

in reactivity between iron and aluminum oxide, over thirty 

model surface structures were examined that might result 

from water physisorption and dissociation by both heterolytic and homolytic mechanisms. The surface energy 

phase diagrams for α-Fe2O3 and α-Al2O3 (0001) indicated 

that the reaction of iron atoms on the surface to form a fully hydroxylated (HO)3Fe-O3-R structure occurs at a water 

vapor pressure threshold many orders of magnitude lower than for aluminum, consistent with experimental observations. Calculations on the models systems also indicated that this hydroxylated surface iron atom can be readily hydrolyzed and solvated when exposed to an aqueous environment. The underlying oxygen layer becomes hydroxylated in 

this process to form a very stable structure that is resistant to subsequent hydrolysis. The relaxations calculated for this 

stable hydroxylated oxygen layer were consistent with the dominant domain observed in the experiments. The minor 

domain is consistent with residual hydroxylated surface iron atoms. 

Hence with the integration of experiment and theory we have successfully identified the surface chemistry that characterizes the fundamental interactions of iron and aluminum oxides with oxygen and water, two of the most prevalent and 

reactive species in the environment. This knowledge of surface structure and reactivity provides a foundation for subsequent investigations into the molecular mechanisms of catalysis, corrosion, and adsorption and transformation of pollutants in groundwater. 

Systematic Validation and Improvement of Quantum Chemistry Methods for the Prediction of Physical and Chemical Properties 

C.A. Gonzalez, R. D. Johnson, and K.K. Irikura (838) 

The use of modern computational chemistry methodologies in the prediction of molecular properties has become increasingly popular mainly due to significant improvements in the algorithms, the accuracy of the methods, and the advent of powerful computer resources. This is particularly true in the area of thermochemistry, where researchers in industry and academia perform quantum chemistry calculations on a routine basis. Recently, there has been an increased 

interest in the use of theoretical predictions of physical and chemical properties made by state-of-the art quantum chemistry methodologies to fill gaps in the available experimental data. This approach could potentially eliminate the cost of 

experimental measurement and will be particularly useful in cases 

where extrapolations based on available experimental data are not possible or are unreliable. However, despite the aforementioned progress, 

the predictive power of most of the quantum chemistry methodologies 

has not been established on solid ground due to the lack of knowledge 

of their relative uncertainties. To understand these uncertainties an 

exhaustive and systematic validation of these methodologies involving 

robust strategies to compare theory and experiment is necessary. In 

addition, the results of such validations must be widely available to the 

scientific community so new scientific discoveries could leverage from 

this knowledge. 

The NIST approach involves the development of state-of-the-art databases in order to disseminate the accuracy, reliability and transferability of the most popular quantum chemistry methodologies in the prediction of physical and chemical 

properties of a large variety of chemical systems ranging from isolated atoms and molecules to condensed phase. The 

theoretical methodologies being studied include semi-empirical Hamiltonians, wavefunction-based ab initio molecular 

orbital theory, and Density Functional Theory (DFT) while the experimental data used in the validation include measurements already available in the literature and also made by researchers at NIST. 

Assessing the Accuracy and Reliability of Density Functional Theory. Density Functional Theory (DFT) has become 

one of the most widely used quantum chemistry methods mainly due to its relatively low computational expense. The 

electronic energy within the DFT formalism is a functional of the electron density that consists of an “exchange” energy 

term and a “correlation” energy term. Although in principle there exists a universal DFT functional that can describe 

the electronic problem exactly, the discovery of such a functional has eluded scientists for years. In practice, arbitrary 

forms for the DFT functionals are parameterized in order to reproduce the experimental energetics of a relatively small 

set of molecular systems. 

T.P. Trainor, A,M. Chaka, P.J. Eng, M. Newville, 

G.A. Waychunas, J.G. Catalano, G. E. Brown, Jr, 

“Structure and Reactivity of the Hydrated Hematite 

(0001) Surface,” Surface Science, 573, (2004). 

A team of NIST researchers is 

working on an integrated approach that will lead to the systematic validation as well as improvements of popular and new 

theoretical methods using high 

quality experimental data. 
34

http://srdata.nist.gov/cccbdb/

CSTL researchers have performed a systematic validation of a particular family of DFT functionals, the so-called “hybrid-GGA”, in order to assess their accuracy and transferability to different chemical properties. The performance of 

these functionals in the prediction of properties such as singlet-triplet gaps, reaction barriers, dipole moments, and polarizabilities showed a marked erratic behavior leading to the conclusion that these functionals are not transferable to the 

computation of different chemical properties. In addition, the results of this work indicate that the major source of error 

can be traced back to the “exchange” functional, which exhibits significant spurious and uncontrollable electron “correlation” energy due to the empirical parameterization used in the development of these functionals. 

Improvements to the Accuracy of Density Functional Theory. 

In order to solve the problems exhibited by the “exchange” 

functionals we proceeded to develop a rigorous and efficient 

method (the SC-α method) for the calculation of the exact 

“exchange” functional. This method has been implemented 

in various popular quantum chemistry software packages and 

it has been tested in more than 40 molecules; the results have 

shown the possibility of computing the exact “exchange” 

functional in complicated polyatomic systems at the computational cost comparable to the inexpensive Hartree-Fock 

formalism. Encouraged by these results, we focused in procedures that could improve the “exchange-correlation” functional as a whole. One of the most difficult steps in the generation of “exchange-correlation” functionals (XC) that do 

not require empirical parameters (and therefore, making it 

more general that the semi-empirical “hybrid-GGA” functionals) is the development of closed forms for the derivatives 

of these functionals with respect to the electron density. 

These derivatives (or potentials as they are known) are critical in the calculation of molecular properties in a “variational” manner and are difficult if not impossible to implement for reliable XC functionals such as the Meta-GGA DFT 

due to their complicated mathematical forms. In this work, we have 

developed a general and simple methodology called the “Approximate 

Self-Consistent Potential” (ASCP) that allows scientists to compute 

the corresponding derivatives without the knowledge of a closed 

form. The method has been implemented in two of the most popular 

quantum chemistry packages and tested in the calculations of absolute 

energies and atomization energies of 20 different molecules. The 

results indicate that the method is robust and sufficiently general. 

Quantifying Uncertainty in Prediction of Enthalpies of Formation and Vibrational Frequencies. It is well recognized 

that a complete expression of experimental measurement includes both the central value and its associated uncertainty. 

The central value alone is incomplete. Throughout computational science, the results of models are usually reported 

without their associated uncertainties, making them incomplete. The term virtual measurement refers to a prediction 

from a computational model together with its associated uncertainty, and emphasizes the analogy with experimental 

(physical) measurement. Our investigation of the uncertainties associated with predictions from quantum chemistry 

models are intended to help “virtual measurements” supplant “calculated results.” The results from ab initio calculations 

differ from experiment by systematic biases, not random errors. For a given ab initio calculation corrections 

can be applied to compensate for this bias and an uncertainty can be determined from the distribution of corrections for a class of molecules. We use the experimental and ab initio data contained in the NIST Computational Chemistry Comparison and Benchmark Database (CCCBDB), which contains data for over 600 molecules, over 100 calculations for each molecule, and over 4000 vibrational frequencies. We combine 

this data with analysis based upon the Guide to the Expression of Uncertainty in 

Measurement, published by the International Organization for Standardization 

(ISO), to determine biases and uncertainties. We have applied this to enthalpies 

of formation and vibrational frequencies. This allows us to upgrade these 

“calculated values” into “virtual measurements”. For the enthalpies of 

formation we found the class of molecules to be critical in determining the bias and uncertainties. For the vibrational frequencies we 

found the uncertainties to be orders of magnitude larger than previously believed. We plan to continue extracting such information from 

the CCCBDB to determine the uncertainties for other calculated 

properties as well. 

I. Gonzalez, C. Gonzalez, V.V. Karasiev, 

E. Ludena, A. Hernandez, "Basis Set 

Dependent SC Alpha Exchange-Only and 

Exchange-Correlation Calculations" J. 

Chem. Phys. 118, 8161, 2003. 

K.K. Irikura, R.D. Johnson III, R.N. 

Kacker, “Uncertainty Associated with 

Virtual Measurements from Computational Quantum Chemistry Models,” 

Metrologia 41, 369-375, 2004. 
35

Theory of Non-Bonded Interactions: Molecular Association and Assembly 

C.A. Gonzalez, V.K. Shen, K.K. Irikura (838), and J.R. Errington (U. of New York, Buffalo) 

Long-range inter-molecular forces play important roles in many chemical and biological systems. They control, among 

others, the base-base interactions leading to the double helical structure of DNA, the function of the special pair in photosynthetic reaction centers, the packing of aromatic crystals, the formation of aggregates, the conformational preferences of polyaromatic macrocycles and chain molecules, as well important bulk fluid properties. It has also been found 

that weak interactions can be important in atmospheric chemistry. For example, the molecule ClOO (chloroperoxyl 

radical, formed by the collisions of the chlorine atoms with oxygen 

molecules) is critical in the catalytic, homogeneous destruction of 

ozone in the stratosphere. Most Cl-O2 collisions lead to excited states 

of ClOO and we have found that many of these excited states surprisingly support weakly bound but stable van der Waals (vdW) complexes 

that must be considered to accurately predict the rates of ozone destruction. 

The molecular systems ideally suited for a detailed study of the intermolecular potentials are vdW dimers and higher clusters of aromatic hydrocarbons formed as a direct consequence of 

intermolecular interactions. Given the maturity reached by quantum chemistry and the improvement in the algorithms 

used in quantum chemical calculations, it is logical to expect that highly correlated ab initio electronic structure methodologies could be a valuable tool to predict the structures and energetics of these clusters. We must assess the validity 

of these methodologies in the case of larger clusters of different aromatic molecules and possibly determine if alternative methodologies that incur lower computational expenses can be devised for these larger systems. Once the reliability and accuracy of these theoretical models are properly validated for gas-phase clusters they can be used for the generation of efficient and reliable empirical force-fields to be used in large-scale simulations of molecular systems in condensed phase such as liquids. This task, of course, also requires robust and efficient simulation methods that predict the 

different physical properties of bulk fluids in a reliable manner. 

Fundamentals of van der Waals Interactions in Aromatic Clusters. The first phase of the project consisted of the use of 

second order Møeller-Plesset perturbation theory, MP2, with different basis sets in order to optimize the geometries and 

compute the relative binding energies of the lowest energy conformers for the dimers, trimers, and tetramers of benzene, 

naphthalene, and anthracene. These systems are a prototype for vdW clusters containing large aromatic molecules 

dominated by a delicate balance between electrostatic and dispersion forces. They were chosen on the basis of the 

availability of reliable experimental data. Overall, the results obtained in this project indicate that the combination of 

molecular dynamics simulations using the MM3 force field, followed by full geometry optimizations at the MP2/6-31G 

level of theory appear to provide a reliable tool for the study of vdW aromatic clusters. One significant outcome of this 

research has been the realization that the use of a small basis sets such as 6-31G with the ab initio MP2 methodology 

predicts binding energies closer to the experimental results than the corresponding binding energies computed with larger basis sets. This surprising result is very important given that calculations of vdW clusters with larger basis sets are 

highly prohibitive due to the computational resources needed. Careful analysis of the data led to the conclusion that the 

source for this interesting behavior is rooted at a fortuitous but systematic cancellation of errors between the lack of convergence of the perturbation method MP2 

and deficiencies in the 6-31G basis set. Given that the study of larger vdW clusters involving more than four aromatic molecules is almost impossible even at 

the MP2/6-31G level, we have performed a systematic comparison of the results 

obtained with the very efficient methodology Hartree-Fock Dispersion (HFD) 

previously developed in our group and the results computed at the MP2/6-31G 

level of theory. It was found that HFD predicts structures (see figures) and binding energies in very good agreement with the MP2/6-31G results. 

The CSTL-led research team develops 

and validates efficient theoretical and 

computational methodologies for the 

description of long-range interactions 

in gas-phase and solution to enable 

prediction of thermodynamic and 

transport properties of fluids. 

Figure shows computed structures of (C10H8)2 and(C14H10)2 

With details in recent publication: 

C. Gonzalez, E. Lim, “Evaluation HFD model as a practical 

tool for probing intermolecular potentials of small aromatic 

clusters: Comparison of the HFD and MP2 intermolecular 

Potentials”, J. Phys. Chem. A 107, (2003).
36

Prediction of Mixture Phase Behavior Using Transition-Matrix Monte Carlo Simulation. The fluid-phase behavior of 

mixtures is a subject of immense industrial and technical importance. For example, in multi-component systems, it is 

the fact that different coexisting phases often have different chemical compositions that serves as the basis for fundamental unit operations, such as absorption, distillation, and liquid extraction. Thus, knowledge of the phase coexistence 

properties of mixtures is crucial to the design of effective separation processes. Because laboratory determination of 

mixture phase equilibria can be potentially expensive and time-consuming, it is highly desirable to have a computational 

means for this purpose. While molecular simulation is ideally suited for this, only within the last twenty years have advances in simulation methodologies made this goal a realistic one. Current state-of-the-art simulation methods directly 

simulate phase coexistence and only yield a single equilibrium point per simulation. Therefore, a large number of these 

simulations are required to obtain phase equilibria data over a range of thermodynamic conditions, which can take a 

substantial amount of time. 

Building upon recently developed transition-matrix Monte Carlo methods, we have developed a new simulation methodology capable of precisely predicting an entire isothermal fluid-phase diagram in a single simulation in a significantly 

shorter amount of CPU time relative to existing methods. To validate the mixture transition-matrix Monte Carlo method 

(M-TMMC), we have investigated a number of binary mixtures whose phase behavior is well known, and also mixtures 

that are known to pose problems for conventional methods. MTMMC produced results in excellent agreement with literature 

data in all cases. Additionally, we found that the relative uncertainties of the predictions were at most 0.2%, an order of magnitude improvement over current methods. Because the method 

determines the system’s free energy as a function of density and 

composition, the utility of the information yielded by this approach goes far beyond that for use in determining phase equilibria, and it should therefore serve as a highly efficient computational tool for studying a wide range of phenomena at the molecular level. 

Integrated Transport Property Program for Key Systems: Data, Models, and Simulation 

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

Efficient design of chemical processing equipment for various industrial 

sectors such as transportation, petroleum refining, energy, and refrigeration requires reliable values for the transport properties viscosity and 

thermal conductivity. The primary approach is the critical assessment of 

all available experimental data and their representation by correlations, 

incorporating theory when possible. Here we have extended the model 

base to the viscosity and thermal conductivity of the linear alkanes noctane, n-nonane, n-decane, and n-dodecane. These are the first such 

models for the heavier alkanes commonly present in liquid-phase fuels. 

The development of these transport properties formulations was driven 

in part by the industrial need/demand for accurate knowledge of the 

properties of hydrocarbon mixtures, including economically important 

fluids such as natural gas and jet fuels. 

The new models represent the viscosity and thermal conductivity surfaces of these compounds over their entire 

fluid region, encompassing the dilute gas to the dense 

fluid and supercritical regions, at temperatures down to 

the triple point. They represent the available experimental 

data to within their experimental uncertainties and extrapolate in a physically meaningful manner. Our models 

for n-octane, n-nonane, n-decane, and n-dodecane have 

been presented in three manuscripts, and at the Properties 

and Phase Equilibria for Product and Process Design 

(PPEPPD) conference in Snowbird, Utah (May 16-21, 

2004). 

The research team is extending this work in 

order to study vdW interactions between polar 

molecules, where dispersion forces and electrostatics interactions might be competitive 

with other forces such as hydrogen bonding. 

In addition, our HFD method will be used to 

probe the dynamics of cluster formation by 

means of Carr-Parinello simulations. 

CSTL researchers pursue an integrated strategy that combines experiment, theory and simulation in 

the development of advanced models for transport properties, leading to predictions of reliable information even when experimental 

data may be lacking. 

M.L. Huber, A. Laesecke, R. Perkins, “Transport 

Properties of n-Dodecane”, Energy & Fuels 18 (2004).

M.L. Huber, A. Laesecke, H.W. Xiang, “Viscosity 

correlations for minor constituent fluids in natural 

gas: n-octane, n-nonane, and n-decane”, Fluid Phase 

Equilibria, 224 (2004). 

M.L. Huber and R.A. Perkins, “Thermal conductivity 

correlations for minor constituent fluids in natural 

gas: n-octane, n-nonane and n-decane”, Fluid Phase 

Equilibria, in press.
37

Complementing our experiment-based approach are computational studies of the properties of systems with model potentials. Such studies can improve fundamental understanding for conditions where measurements are not feasible. Results of high computational accuracy have been published for the shear viscosity, the self-diffusion coefficient, and the 

bulk viscosity.With comprehensive simulation data at over 350 state points, the temperature and density dependences of 

these properties of the Lennard-Jones potential are characterized for the first time over a wide range of fluid states. The 

simulation data for the bulk viscosity has revealed a large critical enhancement similar to that known for the thermal 

conductivity, but extending much 

farther into the supercritical region (it 

can be observed even at 4.5 times the 

critical temperature). From our simulations, the bulk viscosity is now 

known in a wider range of states for 

the Lennard-Jones model fluid than 

for any real fluid. 

The integrated program on transport properties represents a portion of our coordinated efforts on thermophysical properties. These are important to both our infrastructural work (information will be disseminated through such Standard Reference Databases as NIST REFPROP), and to the immediate needs of customers, such as a project on rocket propellant 

RP-1. The ongoing program will continue to focus on both immediate and specific demands for transport property information and on longer-term efforts to improve our predictive capabilities in these areas. 

Thermodynamic Reference Data 

E.W. Lemmon, M.O. McLinden, A.H. Harvey, and R.A. Perkins (838) 

Databases are the primary technology transfer vehicle by which we deliver our collective expertise in fluid properties to our customers. Much of 

our theoretical, modeling, and experimental efforts are directed toward 

improving and extending the databases to meet customer needs. 

New “short-form” equations of state (EOS) for twenty new fluids describe all of the thermodynamic properties using a fixed functional form 

based on the Helmholtz energy. The short fixed form allows us to calculate properties for fluids with limited data sets but with higher accuracies 

(typically 0.1 % to 0.5 % in density, 1 % to 3 % in liquid heat capacities 

and sound speeds) than previously attained for these fluids. REFPROP 

has also been improved by the addition of equations for the dielectric 

constant of both pure fluids and mixtures. 

These new short-form equations of state are based on state-ofthe-art EOS for reference fluids. We have advanced the stateof-the-art for EOS with new formulations for R125 and propane containing new terms and using new fitting techniques 

designed to make the equations more fundamentally sound. 

(Traditional high-accuracy EOS were highly empirical and did 

not have the proper behavior beyond the range of the data, 

while theoretically based EOS had proper qualitative behavior 

but were not of high accuracy.) The new functional form eliminates certain nonphysical behavior in the two-phase region. The addition of nonlinear fitting constraints has yielded an 

equation that achieves proper phase stability, i.e., only one solution exists for phase equilibrium at a given state. We 

have developed new fitting techniques to ensure proper extrapolation of the EOS at low temperatures, in the vapor 

phase at low densities, and at very high temperatures and pressures. 

K. Meier, A. Laesecke, S. Kabelac, “Transport coefficients of the Lennard-Jones model fluid. I. Viscosity”,,J. Chem. Phys., 121(2004) 

K. Meier, A. Laesecke, S. Kabelac, “Transport coefficients of the Lennard-Jones model fluid. II. Self-diffusion”, J. Chem. Phys.121 (2004) 

K. Meier, A. Laesecke, S. Kabelac, “Transport coefficients of the Lennard-Jones model fluid. III. Bulk viscosity”, J. Chem. Phys.122 (2005) 

in press. 

NIST expands the REFPROP 

(Reference Properties) database 

to include over 20 new fluids of 

industrial importance including 

hydrogen sulfide, carbon monoxide, nitrous oxide, toluene, xenon, and R227ea. 

The REFPROP database has been the de 

facto standard in the refrigeration industry 

for many years. NIST was a key player in the 

new ISO standard for refrigerant properties, 

which adopts the same formulations as those 

used in REFPROP. 
38

Development and validation of these high-accuracy 

equations of state require extensive, high-accuracy experimental data, and our recent measurements include 

the heat capacity (CV) and density (p-ρ-T) of propane. 

Between CV and p-ρ-T, our measurements cover the 

entire fluid range from the triple point to 500 K, including low-density gas states and compressed liquid states 

up to 36 MPa. The p-ρ-T data include extensive measurements near the gas-liquid critical point. These data, 

combined with new terms in the EOS, allow us to determine (using only single-phase data) the critical point 

at least as accurately as the available literature values. 

Critical Reference Materials for Mineral Commodities 

J. R. Sieber, A.F. Marlow (839), S. A. Wilson (USGS), and J. T. Wolsiefer, Sr. (Silica Fume Association) 

Mineral wealth has always been a key element of a thriving 

economy. As technology evolves, new mineral resources are 

exploited while traditional industries demand stockpiles of 

ever-improving quality. Thus, the need for mineral Standard 

Reference Materials (SRMs) remains but demands improved 

quality of compositional information. Likewise, the need for 

by-products SRMs is increasing as sustainability and economic pressures push for the transformation of these stockpiles into useful products. 

SRM 1d is the fourth version of an important material. Argillaceous limestone – meaning ‘containing clay’ – is a critical natural resource. Besides its use as a building material, it 

is used to manufacture lime for agricultural and chemical 

processes, cement and concrete, and iron 

and steel. ASTM International Committee 

E01 on Analytical Chemistry of Metals, Ores, and Related Materials, Committee C01 on Cement, 

Committee C07 on Lime and Limestone, Committee C09 on Concrete and Concrete Aggregates, and 

the National Lime Association expressed support for renewal of SRM 1c. Like its predecessors, SRM 

1d will be used extensively for validation of ASTM International and in-house test methods at hundreds 

of laboratories around the world. 

Standard Reference Material 2696 Silica Fume is the culmination of collaboration by the Silica Fume Association and NIST with support from the Federal 

Highway Administration. SRM 2696 is intended primarily for evaluating 

chemical and instrumental methods of analysis of silica fume to satisfy product 

specifications. Silica fume, a by-product of metals refining, metamorphosed 

from industrial waste to an important commodity in the past five to ten years. Certified values were established for silicon (as SiO2) and six other chemical constituents, plus reference values for five constituents and Specific Surface Area 

by nitrogen absorption. All values are products of extensive testing by NIST and collaborating laboratories from manufacturers, distributors, state transportation departments, universities, and commercial laboratories. This allows us to 

leverage their expertise without the investment needed to develop 

it ourselves. 

High-Performance Concrete containing silica fume can have very 

high compressive strength and durability due to low permeability. 

Reduced permeability helps prevent spalling due to the corrosion 

of steel reinforcing bars inside the structure following the infiltration of chlorine from salt. Silica fume is a byproduct of producing 

silicon metal and ferrosilicon alloys. No longer discarded in landCSTL researchers will port the computational engine 

of REFPROP to the Thermo-Data Engine (TDE), a 

new NIST database providing on-demand correlation 

of experimental data. This will give TDE the advantage 

of the inherent thermodynamic consistency of representing properties with equations-of -state versus the 

present approach of representing different properties 

with discrete correlations. Models for aqueous (waterbased) systems and hydrocarbon mixtures containing 

hydrogen and helium will also be developed. 

NIST has long supported US industries that 

rely on mineral resources. 

SRM 1d Argillaceous Limestone exemplifies this 

long-term commitment, versions of SRM 1 have 

been provided by NBS/NIST since 1910. 

SRM 2696 Silica Fume exemplifies our responsiveness to new technology. This SRM was developed to support manufacture and use of this 

high-grade, small particle size silica to improve 

the strength and durability of concrete. 

Both SRM 1d and SRM 2696 were certified using X-ray fluorescence spectrometry

(XRF) with borate fusion sample preparation in the matrix-independent approach 

developed during an Exploratory Research 

project several years ago. 
39

fills, more and more silica fume is being used in concrete instead. Increasingly, state highway and transportation administrations require high-performance concrete in bridges and roadways. In response to the demand and to protect 

against the use of contaminated material, standard-writing organizations around the world are implementing specifications for the chemical composition and physical properties of silica fume. 

Transport Coefficients and Molecular Dynamics 

R. D. Mountain (838) 

Details provided in the Exploratory Research Section of this Report. 

SRM 1c SRM 1d Constituent December 1978 October 2004 

SiO2 6.84 ± 0.16 (2.3 %)b 4.080 ± 0.071 (1.7 %)b

Fe2O3 0.55 ± 0.06 (11 %) 0.3191 ± 0.0068 (2.1 %) 

Al2O3 1.30 ± 0.06 (4.6 %) 0.526 ± 0.013 (2.5 %) 

TiO2

c

 0.07 ± 0.02 (29 %) 0.0306 ± 0.0065 (2.1 %) 

P2O5 0.04 ± 0.02 (50 %) 0.0413 ± 0.0025 (6.1 %) 

MnOd 0.025 ± 0.01 (40 %) 0.0105 ± 0.0003 (2.9 %) 

CaO 50.3 ± 0.6 (1.2 %) 52.85 ± 0.16 (0.3 %) 

SrO 0.030 ± 0.01 (33 %) 0.0303 ± 0.0010 (3.3 %) 

MgO 0.42 ± 0.08 (19 %) 0.301 ± 0.010 (3.3 %) 

Na2O 0.02 ± 0.02 (100 %) 0.0109 ± 0.0016 (1.5 %) 

K2O 0.28 ± 0.02 (7.1 %) 0.1358 ± 0.0046 (3.4 %) 

S Not determined 0.1028 ± 0.0062 (6.0 %) 

ZnO Not determined 0.0022 ± 0.0003 (14 %) 

a

 Due to changes in definitions since 1978, the uncertainties for SRM 1c 

were multiplied by an 

expansion factor of 2 to 

make them comparable 

with SRM 1d. b

 The relative expanded 

uncertainty is given in parentheses. 

c

 This constituent is a reference value for SRM 1d. 

d

 Mn is certified as the 

element in SRM 1d. 
40
41

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. 

Standards Development and Measurements to Support Global Climate Change 

G.C. Rhoderick (839) 

Several species of gases found in the atmosphere that are 

known to contribute to the change in the radiative environment 

of the earth, have been developed as gaseous primary standards (PSMs). These gases are considered greenhouse gases 

and have been monitored throughout the atmospheric environment community worldwide for many years. These gases 

have also gained increased importance since the Kyoto protocol was designed and implemented to promote reduced emissions of greenhouse gases. PSMs were developed previously 

for methane (CH4), carbon dioxide (CO2), tetrafluoromethane 

(CF4), nitrous oxide (N2O), and sulfur hexafluoride (SF6). 

This past year an additional compound, 1,1,1,2-tetrafluoroethane (Halocarbon 134a), was studied and standards developed for the State of California Air Resources Board (CARB). This compound is a replacement for 

halocarbons previously used as refrigerants but being phased out due to their high warming potential. Additionally, new 

PSMs were prepared at atmospheric levels and added to a suite of previously prepared standards. These compounds include carbon tetrachloride, chloroform, trichlorotrifluoroethane (CFC-113), 1,1,1-trichloroethane, and trichloroethylene. 

A total of 22 gravimetrically prepared CH4 in air primary standards now exist and 

are used to define the NIST primary calibration methane scale for laboratory measurements and traceability. The entire suite of primary standards range in concentrations from 0.8 μmol/mol to 10 μmol/mol. Eight of these PSMs also contain nitrous oxide, dichlorodifluoromethane, and 

trichlorofluoromethane. Two PSMs were submitted by NIST for evaluation in a BIPM International Comparison, the 

CCQM-P41 Greenhouse Gas comparison study. One PSM contains methane in air and the second mixture contains carbon dioxide in air. Three PSMs containing methane in air were also compared to methane-in-air PSMs prepared at the 

Climate and Diagnostics Laboratory at NOAA. A suite of three standards containing CF4 and SF6 were prepared to 

value assign the BIPM CCQM-K15 sample. A suite of four standards containing carbon tetrachloride, 1,1,1-

trichloroethane, chloroform, trichlorotrifluoroethane, and trichloroethylene in air and five standards prepared in nitrogen containing the same 

compounds now exist at NIST. This suite of primary standards range in 

concentrations from 5 pmol/mol (ppt) to 100 pmol/mol. The PSMs containing these halogenated species can now be used to analyze real air 

samples as part of the World Meteorological Organization’s International Halocarbon Experiment program. 

Approximately 117,000 US 

companies are engaged in the 

business of environmental 

technologies, with 4,300 of 

those firms exporting internationally. 

Gaseous Primary Standards were developed 

to support: 

• NIST’s in-house standards base for SRM 

certification, 

• international comparison programs, 

• NIST FTIR spectral database project, and 

• International Halocarbon Experiment program sponsored by the World Meteorological Organization, NOAA, and NASA. 

These PSMs will lay the foundation 

for which atmospheric measurements 

can be underpinned and new SRMs be 

developed to support national ambient 

monitoring measurements of those 

species. 
42

Absorption Coefficient Measurements of Aerosol Particle Agglomerates 

C. Presser, C. Sheng (836), and A. Nazarian (SAIC) 

Greenhouse effects associated with climate change may be 

influenced strongly by the chemical and physical properties 

of aerosol particles in the atmosphere. Currently, the largest 

uncertainty in predicting the change in Earth’s global average temperatures over time is due in part to inadequate 

knowledge of the optical properties of atmospheric aerosols, 

such as soot and cloud condensation nuclei. 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 particulate matter (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 characterize the optical behavior of soot PM, 

which combines a laser driven thermal reactor (LDTR), an 

acoustic levitator, and optical pyrometry, as a wellcontrolled thermal environment for the non-intrusive determination of optical, thermal-physical, and chemical-kinetic 

properties. 

Measurements were carried out in the LDTR to determine 

the effect of carbon shape and size on its absorption characteristics. This information is important for characterizing 

particles when their surface is not always spherical and well 

predicted by theory. 

Results were obtained for a spherical and disk-shape 

agglomeration of compacted particles over a range of 

laser heating temperatures. The figure presents the 

effect of heating on the determination of the absorption 

coefficient. The LDTR was modified to include a 

chemical sampling and analysis capability. A sampling 

probe and gas collection arrangement was fabricated 

and operated by vacuum pressure extraction of gases 

into a stainless steel accumulator with a septum. The 

septum allows collection of the gas into a syringe, 

which in turn provides sample for analysis in a gas 

chromatograph/mass spectrometer (GC/MS). The GC 

has a capillary column that allows separation for polycyclic aromatic hydrocarbons (PAHs). PAH identification is of interest since they are considered precursors 

to the formation of soot. The MS (which can scan from 

10 to 800 mass/charge ratios) and NIST MS library are 

used to identify such candidate PAH molecules. 

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 multi-component 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. 

The CSTL-led research team studies the feasibility of a new measurement approach for 

the absorption coefficient of carbon-based 

aerosol particles at high temperature (up to 

1200 K) using controlled laser heating. 

Schematic of Laser-Driven Thermal Reactor

300 350 400 450 500 550 600 650 700 750

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Carbon Absorption of Laser at 1.064mkm

 Carbon Disk Shape

 Carbon Sphere Shape

Absorption Coefficient

Temperature, K

Variation of absorption coefficient with sample temperature for the sphere- and disk-shaped carbon. 

The determination of the optical absorption 

characteristics of aerosol particulates with 

greater accuracy than other existing techniques 

has excellent potential to improve the performance of thermal-optical analyzers, which are 

used extensively to characterize aerosol particles 

that are collected in the atmosphere. 
43

Establishing Measurement Traceability for Gaseous Mercury Emissions Monitoring 

G.D. Mitchell and W.D. Dorko (839) 

The Environmental Protection Agency (EPA) has announced that it will regulate emissions of mercury from coal- andoil fired power plants because of the known health risk posed by environmental exposure. The EPA is focusing its effort on both elemental (Hg 0

) and ionic (Hg++) mercury. Although 

coal-fired power plants are the largest source of mercury emissions 

to the air in the US, mercury is also emitted from other sources such 

as municipal waste combustors, medical incinerators, and hazardous waste combustors. Mercury emitted from power plants and 

other sources is carried by the wind and is eventually deposited 

onto the land and water. Once it enters the water (lakes, rivers, and 

wet lands) it is converted to methylmercury and can enter the food 

chain. 

NIST worked with a commercial specialty gas vendor to procure three sets of gas mixtures of Hg0

 in nitrogen. The 

nominal concentrations of the Hg0

 in the cylinders are 2 μg/m3, 5 μg/m3, 

and 20 μg/m3. Another means of providing gas mixtures for instrument 

calibration purposes is to use a mercury vapor generator. One type of 

generator functions by passing a metered flow of nitrogen over a heated 

pool of mercury. The concentration of 

mercury in the gas stream is calculated 

by knowing the vapor pressure of Hg0

at the thermostated temperature of the 

mercury pool, and the calibrated flow 

rate of the nitrogen. One system of this 

type was also evaluated by NIST by setting the parameters to generate mixtures at the 

same concentration levels as the cylinder gas mixtures. 

The primary analytical instrument used to quantify 

the amount of mercury is a Nippon Instrument 

Corporation Model MA-2000 Mercury Analyzer 

(MA-2), which is equipped with a cold vapor 

atomic absorption (CVAA) detector. The MA-2 is 

an analytical system that can measure mercury in 

discrete liquid or solid samples. A weighed 

amount of a solid or liquid is put into a ceramic 

boat that is then placed in a chamber of the analyzer. The way in which vapor samples are supplied to the MA-2 is to 

pass a measured volume of the gas containing the vapor through an external gold trap to collect the Hg. This gold trap 

is then placed in the MA-2 instrument boat and this is treated as described above for a solid sample. The traps that were 

used to trap the Hg0

 from the gas samples, either from the cylinders or the generator, were tested to determine that there 

was complete collection. The analyzer was calibrated periodically with quantitatively diluted Mercury in Water SRM 

1641d and the cylinders analyzed and delivered to EPA. 

The certified cylinder mixtures are to be used by EPA in their program to audit mercury monitoring sites to determine 

compliance with regulations. Up till this point they had no way by which to provide quality assurance for the audits. 

The program also provided data on the performance of a mercury generation device and these data showed that this 

device is a viable alternate option for calibration and audit of mercury monitors. The 

cylinders containing the 

mercury mixtures are to 

be re-analyzed on a 

periodic basis to determine whether or not there is any degradation of the Hg0

 concentration. Development work has already begun on a 

method for studying ionic mercury (Hg++) to provide traceability for measurements being made in support of EPA’s 

proposed regulations. 

The EPA came to NIST for assistance 

in providing traceability in mercury 

gas standards, which are to be used to 

calibrate monitoring instruments to 

measure mercury to verify compliance 

with EPA emission allowances.

NIST delivered to the EPA three cylinders of mercury in nitrogen whose 

concentrations are certified and 

traceable to the SI units. Also, a report was submitted detailing the findings concerning the performance of 

the mercury gas vapor generator. 

Measured Conc. in 

EPA’s three cylinders 

(2.25 ± 0.08) μg/m3

(5.99 ± 0.18) μg/m3

(22.8 ± 0.68) μg/m3

Comparison between generator output concentrations and measured results 

 Concentration, μg/m3 

 Hg-20 Hg-05 Hg-02

Generator Setting 20.86 5.13 2.48 

Measured on MA-2 21.3 4.97 2.50 

Difference: Measured to Set + 2.1% - 3.1% + 0.8% 
44

Standard Reference Materials (SRMs) and Quality Assurance Activities to Support Measurements of 

Organics on Air Particulate Matter Less Than 2.5 μm (PM2.5) 

S.A. Wise, J.R. Kucklick, B.J. Porter, D.L. Poster, M.M Schantz, R.O. Spatz, and R. Zeisler (839) 

In 1997 the Environmental Protection Agency (EPA) issued new standards for air particulate matter (PM) under the national ambient air 

quality standards including new regulations for PM less than 2.5 μm 

(PM2.5), which is the respirable PM fraction, in addition to the existing 

PM10 standards. Research recommendations have been made by the 

National Research Council at the request of Congress and EPA to focus 

on evaluating what types of particles cause detrimental health effects. 

To support compositional analyses and other investigations on the fine 

PM, quality assurance materials are necessary; however, few appropriate fine particulate materials are currently available to support this research. 

The NIST/EPA agreement has focused on three projects with the goal of providing quality 

assurance and SRMs to support measurements of organic compounds in fine PM including: 

(1) establishment of a interlaboratory comparison program to assess measurement comparability, (2) development of solution SRMs for compounds of interest for PM measurements, and (3) collection of bulk PM2.5 for use as a future SRM. As part of the NIST/EPA 

collaboration, the Organic Speciation Working Group was formed in 2000 to assist in this 

effort by participating in interlaboratory comparison studies and by providing input for the 

development of SRMs to support these measurements. This group has participated in three 

interlaboratory comparison studies for the determination of polycyclic aromatic hydrocarbons (PAHs), nitrated PAHs, alkanes (including hopanes and cholestanes), sterols, carbonyl compounds (ketones and 

aldehydes), acids (alkanoic and resin), phenols, and sugars in PM-related samples. Based in part on the results of these 

studies and input from the Organic Speciation Working Group, priorities for the development of a number of calibration 

solution SRMs were identified including: aliphatic hydrocarbons, PAHs, nitro-substituted PAHs, hopanes/steranes, and 13C-labeled and deuterium-labeled levoglucosan (for use as internal standards). SRM 1494 Aliphatic Hydrocarbons in 

Iso-Octane was issued in early 2004. SRMs 2260a PAHs in Toluene and 1491a Methyl-Substituted PAHs in Toluene, 

which are redesigned solutions with an expanded list of 53 PAHs and alkyl-substituted PAHs, were completed in late 

2004. The remaining solution SRMs are in progress and will be completed in 2005. 

Collection of a 200-g bulk sample of ultra-fine particulate matter at a site in 

Baltimore, MD with a high-volume sampler has been only partially successful. 

Two collections of 20 g each have been used to prepare an interim reference 

material and for distribution in the NIST/EPA interlaboratory comparison exercises for the determination of organic compounds. The results from the second 

interlaboratory study using the interim reference material have been used in 

conjunction with NIST measurements to assign concentration values for the 

compounds of interest. This interim reference material is now available to laboratories involved in EPA PM2.5 research programs for use as a control or reference material. The second PM2.5 material collected in Baltimore has been distributed to over 20 laboratories as part of 

the third NIST/EPA intercomparison study, and results will be available in early 2005. In addition, we are investigating 

alternative approaches to obtaining sufficient quantities of PM2.5 for preparation of an SRM including preparation of a 

fine fraction material from existing PM SRMs and/or other total suspended PM. Additional organic compound classes 

have been identified by EPA for development of additional solution SRMs including additional sugars, saturated and 

unsaturated acids, and quinones.

NIST has entered an agreement with 

the EPA to develop SRMs to support 

analytical measurements of organic 

constituents in PM2.5, to improve interlaboratory comparability of 

measurements, and to provide national measurement traceability. 

The development of SRMs to 

support measurements for organic species in fine PM will 

expand the quality assurance 

capabilities to important source 

markers and species critical to 

human health. 
45

Reference Material (RM) 8785 Air Particulate Matter on Filter Media 

G.A. Klouda (837), J.J. Filliben (ITL), and H.J. Parish (SRI International) 

Combustion aerosol has a major influence on air quality and is known to have a direct and indirect impact on the Earth’s radiative forcing. For the most part, man’s 

activities are linked to these emissions and account for a significant amount of the 

total carbon aerosol found in the atmosphere. To assess impacts and distinguish 

sources of combustion aerosol, numerous methods exist to quantify the amount of 

organic and black (soot) carbon, important in addressing air quality issues and estimating the warming and cooling effects of aerosol on our climate system. However, 

the results vary depending on which analytical method is used, leading to a variety of 

definitions of what constitutes black carbon. 

Researchers in NIST’s CSTL and ITL have collaborated with SRI International 

(Menlo Park, CA) to produce Reference Material (RM) 8785 Air Particulate Matter 

(PM) on Filter Media. RM 8785 was produced by resuspending SRM 1649a Urban 

Dust, sampling its fine fraction (< 2.5 μm aerodynamic diameter) and filtering the 

PM2.5 onto nearly 2000 quartz-fiber filters. Filter ID number and the gravimetrically 

determined mass of fine SRM 1649a uniquely identify each filter. RM 8785 is intended primarily for use in the evaluation of analytical methods used to characterize 

the carbon composition of atmospheric PM2.5 for national ambient air quality standards (NAAQS) monitoring programs. Additionally, RM 8785 will provide the atmospheric chemistry and ocean-sciences community with a 

means to compare methods and laboratories for the measurement of black carbon. 

Through an inter-laboratory and inter-method comparison 

involving NIST, Desert Research Institute (Reno, NV), and 

Sunset Laboratories Inc. (Tigard, OR), concentrations of 

total carbon, black carbon, and organic carbon were measured and values were assigned. Measurements were performed using two widely used thermal-optical protocols: 

one is from the Interagency Monitoring of Protected Visual 

Environments (IMPROVE), and the other is from the 

Speciation Trends Network-National Institute of Occupational Safety and Health (STN-NIOSH). RM 8785 has been assigned a reference value for 

total carbon concentration and information values for black and organic carbon concentrations corresponding to each method. 

Influence of Oxygen on the Shelf Life of Coal Standard Reference Materials (SRMs) 

R. Zeisler (839) and W.D. James (Texas A&M)

Long-term stability of SRMs is essential to customers’ use of the 

materials. CSTL scientists and collaborators validated the stability of 

coal SRMs under proper storage conditions. Oxidative environments 

in combination with elevated temperatures have been found to promote the oxidation of coal samples and lead to changes in composition. 

Initial evidence of change was provided by trace element data provided by instrumental neutron activation analysis (INAA) in SRM 

1632b, which indicated a change in mass fraction values similar to 

the reported carbon change in the material. 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 in a gain of matrix mass, such 

as by irreversible oxidation and therefore increased matrix mass. 

NIST releases RM 8785 

with reference values for 

black and organic carbon 

concentrations 

CSTL researchers and collaborators 

investigate the long-term stability of 

coal samples when users reported a 

change of -1.6% in the measured 

values of the carbon mass fraction of 

the SRM 1632b Trace Elements in 

Coal that was issued in 1985. 

SRI Dust Generation and 

Collection System 
46

The newly developed SRM 1632c Trace Elements in Coal 

(Bituminous) was selected for the study. Twenty-six samples 

each were stored at three conditions: in liquid nitrogen vapor 

and at room temperature in the original containers packaged 

under argon, and for accelerated aging at 50 °C open to air. 

Under proper storage, the samples showed stable mass during 

the more than 4-year observation; only the ones stored open 

at higher temperature gained mass. To directly measure the 

presumed oxidation, a fast (14-MeV) neutron activation 

analysis (NAA) procedure was employed to determine mass 

fractions of oxygen in the coal samples. 

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 %. 

To evaluate the sample stability, test points were set at 6 

months 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. 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. As further 

validation of this gain in sample mass of coal under oxidative storage, an analytical comparison of trace element content in the three storage conditions showed a lowering in 

mass fraction for 22 trace elements corresponding to the gain in sample mass. 

Developing Trace Analytical Methods and Gas Standards for Ammonia in Nitrogen 

T.W. Vetter and W.J. Thorn (839) 

Natural gas turbines used to produce electric power generate significant NOX and SOX pollutants as emissions. Ammonia is added 

to the exhaust stream to neutralize the acid gases forming nitrate 

and sulfate salts, which are collected. The EPA regulations for 

continuous emission monitors (CEMs) require that ammonia not 

exceed 3 μmol/mol excess in the stack emissions. Similarly, alternative fuels such as compressed natural gas (CNG) and hydrogen 

can generate ammonia in mobile source exhaust (cars and trucks), 

which must be monitored. Currently, there are no reference materials available. 

Preliminary measurements were made this year evaluating three primary methods on a series of ten ammonia working 

standards from 5 μmol/mol to 640 μmol/mol. Ammonia concentrations for two of the working standards (40 μmol/mol 

and 160 μmol/mol) were determined by a classical titrimetric method with relative expanded uncertainties of less than 

1 %. All of the working standards were measured during an evaluation of a commercial ammonia instrument, which 

catalytically oxidizes ammonia to nitric oxide (>95 % efficiency) and then measures the nitric oxide by chemiluminesence. This method is of high interest because of the availability of a large quantity of nitric oxide primary standards 

in support of NIST SRMs. Additionally, all of the working standards were measured by fourier transform infrared spectroscopy (FT-IR), and a non-linear regression plot of the measured absorbances vs. concentration was developed. NIST 

has compared the ammonia values measured by the three analytical techniques and found that the 2 % to 4 % differences between the methods are difficult to resolve because the ten working standards of ammonia are decaying in concentration over time compared to their gravimetric preparation values. 

-1

0

1

2

3

4

0 10 20 30 40 50 60

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"

Change in mass

calculated from measured

change in oxygen fraction

Gravimetry 

The measurements provided by NIST give assurance that properly packaged coal SRMs, protected from oxygen and heat, are stable during 

their specified shelf life. Gravimetric monitoring 

of coal SRMs during storage should be employed 

by NIST and customers alike as a cost-effective 

quality measure. 

CSTL is developing a primary method 

for assigning ammonia concentrations to 

gas standards in the 1.0 μmol/mol to 

100 μmol/mol range. These standards 

are of interest to the EPA for mobileand stationary-source monitoring; and 

of interest to specialty gas mixture vendors who service these applications.
47

NIST ammonia in nitrogen working standards: NIST is working with BOC Gases to produce ammonia-in-nitrogen calibration gas standards by gravimetric dilution. For the first batch of 14 standards, FT-IR spectroscopy was used to 

measure ammonia absorbances as a function of the ammonia concentration and results indicated a non-linear FT-IR 

response curve for some of the cylinders. Work to produce a suitable suite of working standards is continuing. 

Titration primary method for determining ammonia: The ammonia concentration was determined in two of the working 

standards whose nominal levels were at 40 μmol/mol and 160 μmol/mol. The sample gas was metered into a solution 

of excess boric acid to form ammonium borate. The ammonium borate was neutralized by back titration with standardized hydrochloric acid to an original pH endpoint of the of the boric acid solution. The titrimetrically-determined mole 

fraction of ammonia in the two mixtures is (37.5 ± 0.3) μmol/mol and (151.5 ± 0.8) μmol/mol. 

Oxidation of ammonia to nitric oxide and chemiluminesence analysis: NIST evaluated a commercial instrument, which 

oxidizes ammonia to nitric oxide and then measures the chemiluminescence response. The two highest working standards were diluted using a commercial mass flow dilution system to generate ammonia reference mixtures similar to 

sample working standards being analyzed. Each of the working standards was assigned a concentration value, which 

was within 95 % of a similar NO response. The corresponding values for the two mixtures above are 

(39.1 ± 0.8) μmol/mol and (155.7 ± 0.8) μmol/mol. All of the measured values are observed to be 4 % to 6 % below the 

gravimetric blend values; suggesting that these working standards are unstable. Using the ammonia chemiluminescence 

instrument, we will attempt to link ammonia concentration determination to our primary nitric oxide gas standards. 

Work will also continue on the titrimetric method to provide direct linkage to the SI. Permeation systems will also be 

investigated in order to provide the conversion efficiency parameter for the chemiluminescence instrument. 

New Standard Reference Material (SRM) for Organic Contaminants in House Dust 

M.M. Schantz, J.M. Keller, J.R. Kucklick, D.L. Poster, H.M. Stapleton, S. Vander Pol, and S.A. Wise (839) 

House dust is a repository of pesticides and other chemicals used indoors or tracked in from outdoors, and as such can 

be used as an indicator of indoor exposure. Pesticides associate with house dust primarily through interior use of pest 

control formulations, vapor intrusion from foundation and 

crawl space treatments, and tracking-in of lawn and garden chemicals. PAHs derive from indoor sources such as 

combustion, cooking, and smoking, and from track-in of 

contaminated yard soil or residues from garage floors. 

PBDEs are commonly used flame retardant compounds 

added to many plastics, resins, and textiles which are then 

incorporated into products including TVs, computers, 

furniture, and carpets. NIST is developing a house-dust 

reference material with values assigned for a wide range 

of organic contaminants for quality control in these measurements. 

Over 100 contaminants, including pesticides, PAHs, 

PCB congeners, and PBDE congeners, will be value 

assigned in SRM 2585 using an approach similar to 

that used for other natural-matrix reference materials, such as mussel tissue and sediment. Three 

methods were used to quantify a number of pesticides, PAHs, and PCB congeners in the house dust. 

Using one of the methods, the material was found to 

be homogenous at a 1 g sample size. Cis- and transchlordane are present in the material at concentrations between 150 μg/kg and 250 μg/kg. Fluoranthene and pyrene are 

present in the material at concentrations between 3000 μg/kg and 4500 μg/kg. The individual PCB congeners are lower 

in concentration (<50 μg/kg for each congener). Two methods were used to value assign the PBDE congeners in SRM 

2585. PBDE 209 was the dominant PBDE congener in SRM 2585 at a concentration of 2600 μg/kg. 

NIST has produced SRM 2583 and SRM 2584 

for lead and other trace elements in a house dust 

matrix. SRM 2585 Organic Contaminants in 

House Dust will be issued with concentration 

values assigned for pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated 

biphenyl (PCB) congeners, and polybrominated 

diphenyl ether (PBDE) congeners. 

The house dust SRM 2585 will provide a control sample 

for laboratories routinely monitoring indoor dust. With 

the increased interest in the PBDE concentrations in all 

types of environmental samples, the assignment of concentrations for this group of compounds in many different matrices, particularly those associated with human 

exposure, will be important. 
48

Method Development and Measurements of Polybrominated Diphenyl Ethers (PBDEs) in Tissue, 

Serum, and Sediment Standard Reference Materials (SRMs) 

H.M. Stapleton, J.M. Keller, M.M. Schantz, and S.A. Wise (839) 

Polybrominated diphenyl ethers (PBDEs) are flame-retardant compounds that are commonly added to many plastics, resins, and textiles 

that are then incorporated into products such as TVs, computers, furniture, and carpets. PBDEs can volatize or leach out of the products in 

which they are applied and be transported long distances in the environment, due to their physico-chemical properties. PBDEs are now 

considered ubiquitous environmental contaminants and much attention 

has been focused on their transport, uptake, and fate in both humans 

and the environment. Presently, there is an increased need for measurements of PBDEs in environmental matrices and 

human serum. SRMs with certified concentrations for individual PBDE congeners are currently unavailable, and they 

are needed to ensure quality control for these measurements. 

Ten existing natural-matrix SRMs have been analyzed for determination 

of a suite of 26 PBDE congeners. These SRMs include marine mammal 

tissue, fish tissue, mussel tissue, human serum, marine sediment, and 

house dust. The GC/MS method, which uses on-column cool injection, 

has allowed for the measurement of the fully brominated PBDE congener (2,2’,3,3’,4,4’,5,5’,6,6’-decabromodiphenyl ether; BDE 209) using 

an isotope dilution quantification approach. This congener has often 

been difficult to measure due to its thermal instability. BDE 209 was 

observed to be the dominant PBDE congener in the sediment and housedust SRMs, and thus, these SRMs will be useful for laboratories interested in routine measurements for BDE 209. 

Other flame retardants such as hexabromocyclododecane (HBCD) are now being used at higher rates as a replacement 

for PBDEs in some parts of the world. Thus, there is interest in the measurements of HBCD in environmental samples. 

Additionally, evidence suggests that PBDEs may be metabolized by some organisms to hydroxylated and methoxylated 

derivatives. Preliminary evidence suggests that methoxylated PBDEs (MeOBDEs) are present in the marine mammal 

blubber and cod liver oil SRMs. Measurements are planned for HBCD and MeOBDEs in these environmental-matrix 

SRMs to provide reference values for these new flame-retardant-related compounds. 

Standard Reference Materials (SRMs) for Contaminants in Marine Tissue and Sediment 

S.J. Christopher, R.D. Day, W.C. Davis, S.E. Long, R.S. Pugh, M.M. Schantz, J.R. Kucklick, D.L. Poster, J.M. 

Keller, H.M. Stapleton, K.E. Sharpless, C.S. Phinney, B. J. Porter, E.A. Mackey, R.O. Spatz, C.E. Bryan, J.R. 

Sieber, R. Zeisler, S.A. Wise, and G.C. Turk (839) 

Targeted environmental monitoring efforts, regulatory pressures, 

and compounds of emerging concern are expanding the list of 

potentially toxic contaminants that impact marine systems and 

challenging the analytical community to develop new measurement technologies for organic pollutants and trace metals. NIST 

is responding by providing marine-matrix SRMs to the marine 

environmental research community for method validation and 

quality assurance purposes and is at the forefront of developing 

and disseminating new analytical technologies for measuring 

contaminants in marine reference materials. 

The value assignment of SRM 1947 Lake Michigan Fish Tissue highlights the collaborative effort undertaken to provide a marine reference material with a comprehensive suite of contaminant values, as well as information on nutritive properties. 

Several classes of compounds are represented in this material, ranging from selected 

trace elements, including total mercury and methylmercury, and organic contamiNIST researchers develop a gas 

chromatography/mass spectrometry 

(GC/MS) method quantify the flameretardants PBDEs in a variety of existing natural matrix SRMs. 

Legislation in the US is now being 

considered to reduce the use of PBDEs 

in consumer products. Numerous 

corporations that produce PBDEladen products will be affected. These 

companies have expressed interest in 

having SRMs to test their products to 

ensure that they conform to these 

regulations. 

New classes of organic and organometallic 

contaminants have been certified in various marine tissue and sediment SRMs. 

New analytical approaches have been developed to certify trace elements fish tissue 

and in SRM 2703 Marine Sediment for 

Microsampling. 
49

nants, including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, and polybrominated diphenyl ether 

(PBDE) congeners to proximates and fatty acids. New methods were developed to measure compounds like PBDEs, 

which are flame-retardant compounds added to consumer plastic products. These compounds are chemically similar to 

PCBs, with levels increasing in the US population. PBDEs are currently being measured in two marine sediment 

SRMs (1941b Organics in Marine Sediment and 1944 NY/NJ Waterway Sediment) and three mussel tissue SRMs 

(1974b, 2977, and 2978). All PBDE measurements required the preparation of custom primary standards from chemically pure solutions and solids, reduction of background levels in the analytical blank, and development of gas chromatography mass spectrometry (GC/MS) methods to accommodate these thermally labile compounds. 

Significant effort went into development of new complementary strategies for determination of organometallic species 

in marine tissue SRMs. For example, microwave-assisted acid extraction was used in combination with solid-phase 

microextraction gas chromatography-inductively coupled plasma mass spectrometry (SPME-GC-ICPMS) to measure 

organic and inorganic fractions of Hg and organotin compounds in SRMs 1947, 1946 (Lake Superior Fish Tissue), and 

bivalve tissues, SRM 1566b Oyster Tissue, SRM 2976 Mussel Tissue (Trace Elements and Methylmercury), and SRM 

2977 Organics in Mussel Tissue. Some of these SRMs were also screened for As and Se content using liquid chromatography coupled to collision cell ICPMS, with the goal being to eventually provide concentration values to SRM customers tasked with measuring speciated forms of metals in various environmental matrices. 

A new certification strategy was tested and implemented for determination of trace elements in SRM 1947 that is applicable to all homogeneous reference materials. Where multiple jars of a homogeneous NIST SRM need to be analyzed, 

a new standard additions/internal standard procedure was created to streamline certification measurement efforts. In the 

new procedure, multiple (typically 6) samples of the candidate certified reference material are accurately weighed into 

digestion vessels and gravimetrically spiked with an internal standard solution. Increasing amounts of a multi-element 

spike solution are then gravimetrically added to each vessel, ranging from no spike to a high spike, to effect a standard 

additions calibration curve. From this point on all measurements of 

the digested samples will be based on analyte/internal standard 

ratios, and all sample handling, including removal of the solutions 

from the digestion vessels and any subsequent dilutions, does not 

need to be done quantitatively. This is one of the main benefits of 

this approach. The other benefit is that we do not have to perform 

sample splits on each sample (SRM jar) to be measured. Values 

and uncertainties are calculated by linear regression of the measured analyte/internal standard ratios vs. the amount of standard added, and the slope of the standard additions curve can 

be used to predict concentration values in similar matrix controls run concurrently during experiments. 

This research group is also developing a new marine sediment standard, SRM 2703. The SRM material was collected at 

the mouth of the Baltimore Harbor, and is primarily intended for use in evaluating analytical methods for the direct determination of selected elements in solid samples of marine or fresh water sediment and similar matrices. SRM 2703 

was specifically developed for microsampling techniques. Direct and slurry sampling, as well as dissolution techniques 

using typically milligram size samples can employ this SRM to benchmark their procedures. A thorough homogeneity 

assessment of the material was conducted using instrumental neutron activation analysis (INAA), laser ablation inductively coupled mass spectrometry (LA-ICPMS), micro-beam X-ray fluorescence (μXRF), and micro proton beam induced X-ray emission (μPIXE) techniques complemented the INAA assay with homogeneity results for significantly 

smaller sample sizes. 

S T i

Fe K 

low

high

S T i

Fe K 

low

high

S T i

Fe K 

low

high

Micro-PIXE images of SRM 2703. Sample 

were analyzed using a 3 MeV focused proton 

beam. Images were collected for all elements 

found in the PIXE spectrum. The total scan 

size of 750 μm x 750 μm was divided into 8 

equal regions, each being quantitatively evaluated. Only sulfur showed heterogeneity directly observable from the 2D maps in all 

SRM samples. Detailed analysis of the spectra 

from 8 smaller regions revealed homogeneity 

of SRM 2703 for Al, Si, Cl, K, Ti, Mn, and Fe 

at the probed 1 μg to 7 μg sample size. 

Analysis performed by a research team at Rudjer Boskovic Institute in Croatia. 

A new, more efficient, certification strategy that utilizes an internal standard produces standard addition curves that can 

be used to predict concentration values 

and does not require quantitative dilutions to produce quantitative results. 
50

Additional measurements with several solid sampling procedures and dissolution procedures were provided by collaborating laboratories with the following techniques: atomic absorption spectrometry (AAS), inductively coupled plasma 

atomic emission spectrometry (ICPAES), and ICPMS, and X-ray fluorescence (XRF). All assays were designed to establish communicability of values between those conventionally certified in SRM 2702, representing the parent material, and the SRM 2703 measured at small sample sizes. These measurements confirmed that the composition of the 

material had not changed in processing and that the measured values in SRM 2702 can be utilized for value assignment 

of SRM 2703. 

We will continue to develop analytical methods to measure environmental compounds of emerging concern in current 

issue and future marine tissue and sediment SRMs, including perfluorooctanesulfonate (PFOS), perfluorooctanoic acid 

(PFOA), other types of brominated flame retardants, classes of compounds like phthalate esters and their metabolites, 

which are prevalent in plasticizers, and representative compounds comprising the range of pharmaceutical and personal 

care products. We will also measure methylmercury and organotin compounds in marine sediment and tissue SRMs. 

Marine Environmental Specimen Bank Activities

P.R. Becker, R.S. Pugh, M.B. Ellisor, J.R. Kucklick, S.J. Christopher, S.S. Vander Pol, R. Day, J.M. Keller, 

C. Bryan, H.M. Stapleton, and S.A. Wise (839) 

Environmental specimen banking is the long-term preservation of 

representative environmental specimens for deferred analysis and 

evaluation. A systematic well-designed specimen bank program is 

not only a valuable component of real-time monitoring and basic 

research, but it also enables investigators to extend their research 

into the past and provides for future verification of analytical results. Formal environmental specimen banks are recognized internationally as integral parts of long-term environmental monitoring 

and research. There are two national environmental banking systems in the US, each having a different purpose. One is known as 

CASPIRTM, the Centers for Disease Control and Prevention (CDC) 

and the Agency for Toxic Substances and Disease Registry 

(ATSDR) Specimen Packaging, Inventory and Repository. CASPIR cryogenically archives specimens for national public health 

investigations. The second is NIST’s National Biomonitoring Specimen Bank (NBSB) that cryogenically archives 

specimens for environmental research. Both specimen banks include well-developed banking protocols, computerized 

sample tracking (chain-of-custody) systems, maintenance of many forms of data associated with original specimens, 

and large investments in state-of-the-art facilities and equipment required to store specimens over relatively long periods of time. Both programs have emphasized cryogenic storage, using ultra-cold (-80 °C) electric freezers and liquid 

nitrogen vapor storage (-150 °C), security systems, and monitoring of storage conditions 24 hours a day, 365 days a 

year. 

The NBSB was originally established in 1979 by NIST at its Center 

for Neutron Research. In 2002, a NBSB satellite facility was established by NIST at the Hollings Marine Laboratory (HML) in Charleston, SC. This facility, the Marine Environmental Specimen Bank (Marine ESB) is devoted to the cryogenic banking of environmental 

specimens collected as part of ongoing research and monitoring programs conducted by other agencies, such as the National Oceanic and 

Atmospheric Administration (NOAA) and the U.S. Department of the 

Interior (DOI), in the marine and coastal environment of the US. 

Although the banking of marine specimens has always been a major 

part of the NBSB (banking of mussels and oysters for NOAA’s Mussel Watch Program, sediments and fish tissues for NOAA’s National 

Status and Trends Program, and marine mammal tissues for NOAA and DOI), the establishment of the Marine ESB at 

the Hollings Marine Laboratory in association with NOAA, two institutions of higher learning and research, and a State 

marine research laboratory, has provided major resources and support for expansion of specimen banking nation-wide. 

NIST is expanding its environmental 

specimen banking activities through the 

recent development of the Marine Environmental Specimen Bank at the Hollings Marine Laboratory. With the increased interest in newly emerging contaminants, the Marine ESB will be a 

valuable resource for establishing temporal trends for these compounds in marine ecosystems. 
51

The National Marine Mammal Tissue Bank, which was established by Federal legislation in 1992, is maintained by 

NIST for the National Marine Fisheries Service and the Fish and Wildlife Service as a component of the NBSB and 

Marine ESB, with the Marine ESB providing the lead. Presently, NIST maintains 2,504 tissue samples collected from 

885 individuals representing 37 species of marine mammals from throughout the US coastal waters, including Alaska. 

These samples are collected from strandings, animals taken incidentally in fishing operations, and animals harvested by 

Alaska Natives for food. Since the establishment of the Marine ESB, NIST has used its cryogenic banking expertise to 

develop protocols and to collect and archive blood and blubber samples for NOAA’s ongoing bottlenose dolphin health 

assessment studies, eggs collected as part of a DOI environmental monitoring program on Alaska seabird colonies, and 

eggs and feathers collected as part of a DOI peregrine falcon monitoring program. To date, blood and blubber have 

been collected from 215 bottlenose dolphins, 434 eggs have been 

archived from five species of arctic seabirds throughout Alaska as 

have 9 eggs and 26 feathers from the peregrine falcon program. 

The primary function of the Marine ESB is to provide samples for 

retrospective analysis. In 2004, aliquots of blubber specimens collected from California sea lions during 1993 to 2002 and banked in 

the NMMTB were analyzed by NIST for polybrominated diphenyl 

ethers (PBDEs) and hexabromocyclododecane (HBCD), which are 

synthetic anthropogenic flame retardants that have now become 

ubiquitous contaminants; temporal trends have indicated concentrations are increasing rapidly in many matrices. The analysis of these 

banked samples was to determine if temporal trends for these compounds are also discernable in this species of marine mammal that is 

common on the west coast of the United States. The results indicate a significant temporal trend with HBCD with a 

doubling time of 2 years. The specimens archived in the Marine ESB will provide a valuable resource for investigating 

temporal environmental trends in these new compounds and for determining patterns of past exposure in marine biota. 

NIST will continue to work with the HML partners 

and other Federal agencies and partners to expand 

environmental specimen banking. NIST staff are 

presently working with these partners to add banking 

as part of a future sea turtle health assessment program, to re-institute banking as part of the Mussel 

Watch Program, to establish a genetic cryo-bank as 

part of a National Genetics Archive initiative for 

marine biota, and to expand the banking of bird 

specimens to include other kinds of species and types 

of tissue samples on a national and international 

level. And finally, existing specimens being held by 

the bank are being identified for future work on identifying and measuring new contaminants of interest 

as related to issues on ocean and human health. 

NIST Aids NOAA with Bottlenose Dolphin Health Assessments 

J.R. Kucklick, S.J. Christopher, P.R. Becker, R.S. Pugh, M. Ellisor, J.M. Keller, 

C.E. Bryan, and J.E. Yordy (839) 

Since 2000, NOAA has studied the health of bottlenose dolphins by collecting data and samples from animals captured and then released at locations along the US Atlantic and Gulf Coasts. Bottlenose dolphins accumulate persistent organic pollutants, such as polychlorinated biphenyls 

(PCBs) and brominated flame-retardants to some of the highest observed 

levels in wildlife, hence they are at risk from the toxic effects of these 

compounds. For instance, in Sarasota Bay, where sampling occurs twice 

per year, there is nearly complete mortality of dolphin calves born to firsttime mothers. It is hypothesized that persistent organic pollutants offloaded from the mother via milk play a major role in this reproductive 

failure. In 2002 NOAA asked NIST to provide technical assistance for the 

The establishment of the Marine ESB in association 

with the HML has provided a renewed interest in 

specimen banking as part of marine environmental 

monitoring and research. With the recognition that 

newly emerging compounds are appearing in the 

environment with potential environmental and 

health effects, the value of the specimens presently 

held by the bank for determining past exposure patterns and for evaluating temporal trends in concentrations of these compounds is becoming more apparent. 

NIST data help provide a basic 

understanding of contaminant 

burdens in bottlenose dolphins 

and provide exposure data for 

toxicological studies performed on 

the animals. 
52

project by (1) designing a protocol for collecting, handling, and storing blood, blubber, and skin to be used for organic 

contaminant and/or trace element analyses, (2) permanently archiving blood and blubber samples for future study, (3) 

analyzing persistent organic pollutants and trace elements in samples collected during dolphin health assessments, and 

(4) providing technical assistance in the field during sample collection. 

NIST developed a detailed protocol for collecting samples from bottlenose dolphins 

during health assessments. The protocol covers the collection of skin, plasma, whole 

blood, and blubber samples to be analyzed for trace elements (including mercury), 

persistent organic pollutants, and perfluorinated compounds, such as perfluorooctane 

sulfonate (PFOS). Since 2002 NIST personnel have successfully applied the collection protocol during 11 health assessments from 4 locations resulting in samples from 

over 150 dolphins. Seven additional health assessments are scheduled for fiscal year 

2005. Subsamples of plasma and blubber are being archived in the Marine Environmental Specimen Bank. Blubber and blood samples are being analyzed for over 100 

individual persistent organic pollutants including brominated flame-retardants. Blubber samples from 22 mother-calf 

pairs were analyzed revealing that the majority of the persistent organic pollutant load is passed to the calf. Milk samples collected from the mother dolphins and blood from mother 

and calf dolphins are currently being examined to better understand contaminant transfer from milk, especially for the brominated flame retardant compounds. Trace elements including 

mercury were determined in blood samples from dolphins 

sampled in Sarasota Bay, Florida and coastal New Jersey. A 

technique has also been developed to measure mercury in skin 

biopsy samples. This may allow the use of skin collected from 

remote dart biopsies to be used for the assessment of mercury. 

NIST will continue to work with NOAA as they expand their 

dolphin health assessment project to other areas along the coastal US. Work is underway to make use of highthroughput techniques for the analysis for persistent organic pollutants in blood and blubber. The target list will be expanded to include other types of compounds of emerging concern, such as additional flame retardants and perfluorinated 

compounds. A control material for dolphin blood will be developed and value assigned to provide a benchmark for 

blood analysis. 

Interlaboratory Comparison Exercises for Organic Contaminants and Trace Elements in Marine 

Mammal Tissues 

J.R. Kucklick, S.J. Christopher, P.R. Becker, R.S. Pugh, M.B. Ellisor, S.S. Vander Pol, E.A. Mackey, R.O. Spatz, 

B.J. Porter, M.M. Schantz, G.C. Turk, and S.A. Wise (839) 

Laboratories measuring contaminants in the marine environment must 

assess the accuracy and precision of their measurements. Quality control 

of measurements made on marine environmental samples is vital to the 

accurate assessment of marine pollution and its effects on wildlife and 

human health. Representative control materials needed to benchmark 

analytical methods and measurements are often limited or not available 

for many marine matrices of interest (e.g., marine mammal tissues and 

marine fishes). Consequently, analysis may be undertaken on samples without control materials that are similar in nature to the sample. To help address this need, NIST along with other US Government sponsors, initiated several programs to assess the data quality of laboratories performing chemical measurements on marine-related samples. 

The first such program for environmental measurements was initiated in 1987 and funded in part 

until 2000 by the National Oceanic and Atmospheric Administration’s (NOAA’s) National 

Status and Trends Marine Monitoring Program (NOAA/NS&T). This program provides mechanisms for assessing the interlaboratory and temporal measurement comparability of polycyclic 

aromatic hydrocarbons, polychlorinated biphenyl (PCB) congeners, and chlorinated pesticides in 

bivalve mollusk, sediment, and fish samples. The program includes components for developing 

improved analytical methods, producing NIST Standard Reference Materials (SRMs) and other 

Analytical work completed for NOAA ties 

measurements to a national metrology laboratory with a history of providing highquality measurement data to NOAA. Since 

the dolphin health assessments are longterm projects, NIST’s involvement will provide continuity in the analytical data generated on the project. 

NIST helps benchmark and improve the quality of analytical data 

on the marine environment by 

conducting interlaboratory comparison exercise programs. 
53

control materials, conducting annual interlaboratory comparison exercises, and coordinating workshops to discuss exercise results. This program continues as the NIST Intercomparison Program for Organic Contaminants in the Marine 

Environment with partial support from fees paid by the participants, and it served as the model for the Interlaboratory 

Comparison Exercises Program for Organic Contaminants and Trace Elements in Marine Mammal Tissues. 

NIST expanded the Interlaboratory Comparison Exercise for Organics in Marine Mammal Tissues in 1999; and the trace element complement to the exercise was formalized in 2000. Since this time the 

participation has increased dramatically. For example, the number of 

laboratories participating in the organic exercise increased from 10 in 

1999 to 25 in 2003. The participants in the exercises are international, with nine countries involved in the trace element exercise and 

six in the organic exercise in 2003. Results from the program led to 

the development of a suite of control materials for trace elements in 

marine mammal tissues and the production of one Standard Reference Material, SRM 1945 Organics in Whale Blubber. The organics exercise has been successful as a source of data on 

constituents that were not certified in SRM 1945; for example, fatty acids and brominated flame retardants were determined in this SRM by six of the participating laboratories. The trace element exercise has led to the development of 

three trace element control materials from marine mammal livers, a pilot whale liver homogenate in 1991, and beluga 

whale and pygmy sperm whale liver homogenates in 1997 

and 2003, respectively. The trace element exercise also 

used an innovative approach to arrive at a consensus mean 

and uncertainty data using a maximum likelihood solution 

model developed in NIST’s Information Technology 

Laboratory. 

The SRMs and control materials developed through this 

program and the interlaboratory comparison exercises are 

mandated for use by researchers in the US who are funded by NOAA to perform measurements of trace elements and 

organic contaminants in marine mammal tissues. NIST will continue to administer and coordinate interlaboratory comparison exercises for chemical analysis of marine mammal tissues. Future exercises will ask participants for compounds 

of emerging interest such as brominated and fluorinated compounds, toxaphene congeners, and organometal contaminants, such as butyl tins and methylmercury. The next round of interlaboratory comparisons will occur in 2005. Also 

in 2005, the data compiled from the exercises using SRM 1945 as a control will be used to assist in updating the Certificate of Analysis with values for additional polychlorinated biphenyl congeners, brominated diphenyl ether congeners, 

toxaphene, and fatty acids. 

Detection of Trace Anthropogenic Contamination in Aquatic Ecosystems using Fluorescence 

Excitation-Emission Matrix Spectroscopy 

R.D. Holbrook (837) and P.C. DeRose (839) 

Aquatic resources play a key role in supporting and maintaining human activity. Aquatic systems provide potable water 

supplies to meet growing drinking and agricultural water requirements, and are used as receiving streams in the disposal 

of treated wastewater effluent. However, the nearly ubiquitous 

presence of trace organic contaminants in receiving streams has 

prompted worldwide concern about their impact on human 

health, largely due to the documented sexual disruption of aquatic 

wildlife caused by some trace contaminants as documented in the 

literature (for example, Environmental Science and Technology, 

2003, 37, 1142-1149). Current analytical procedures for measuring trace organic contaminants in aqueous samples are both expensive and laborious, indicating the need for a sensitive screening method that is capable of detecting organic compounds that 

originate from biological wastewater treatment systems. 

The high frequency of contaminant detection in aquatic systems and extremely low biologically-active concentration 

(i.e., ng/L) recently led the National Research Council to recommend better monitoring of receiving streams. A simpliIndependent analytical methods at 

NIST (inductively coupled plasma 

mass spectrometry and instrumental 

neutron activation analysis) produced 

data to provide a reliable benchmark 

to assess the performance of participating laboratories. 

Results: 

Comparability of measurements for organic contaminants has improved for those laboratories 

with a longer history of participation in the exercises, e.g., those that have participated since 1999 

have improved their accuracy by 10% and their 

precision by 20%. 

CSTL scientists investigate the use of fluorescence excitation-emission matrix spectroscopy, or EEM, as an applicable means 

of distinguishing treated wastewater effluent 

from “uncontaminated” receiving water 

samples. 
54

fied screening method would facilitate a wider range of samples/environments being examined. It is expected that environmental practitioners would be the immediate beneficiaries, followed by the general public, if a link between these 

compounds and human health is established. 

A method of EEM collection and data analysis was developed using samples 

collected upstream and downstream of an advanced wastewater treatment facility. The EEM procedure took approximately 20 minutes per sample, required 

little sample volume (< 10 mL) and few preparation requirements. Data analysis was adopted from the article of Chen et al (Environmental Science and 

Technology, 2003, 37, 5701- 5710). A unique fluorescence distribution was observed for the downstream samples as shown in the figure, indicating that the 

presence of treated effluent could be detected using EEM. Specifically, a significant increase in fluorescence intensity was observed in region 1 (aromatic 

proteins) at excitation wavelength between 230 nm and 260 nm and emission 

wavelength between 300 nm and 340 nm as seen in the table. 

Typical EEM contour plots of upstream (left) and downstream (right) samples. The red lines are the superimposed regions that 

are described in the table. Note the increase in fluorescence intensity in Region I (lower left area) between the upstream and

downstream samples. Each contour line represents 1/20 of the maximum fluorescence intensity. 

Excitation-Emission Region Description and Results of Regional Integration of Upstream and Downstream Samples 

 

 Organic Matter Type Excitation Emission 

Portion of Total Fluorescence Signal 

(%) 

 Wavelength (nm) Wavelength (nm) Upstream (n = 6) Downstream (n = 15)

Region I Aromatic Proteins 230 to 260 300 to 340 9.9 ± 0.3 13.5 ± 2.3 

Region II Aromatic Proteins 230 to 260 340 to 380 25.5 ± 0.8 26.3 ± 1.3 

Region III Fulvic Acids 230 to 260 380 to 500 42.5 ± 0.5 38.7 ± 3.1 

Region IV Soluble Microbial Products 260 to 400 300 to 380 8.8 ± 0.1 9.4 ± 0.6 

Region V Humic Acids 260 to 400 380 to 500 13.3 ± 0.4 11.9 ± 0.9 

Results are average ± 1 standard deviation 

Results: 

The signature fluorescence distribution of downstream samples is 

noteworthy due to the extensive 

treatment processes employed at the 

advanced wastewater treatment 

facility. The fluorescence distribution of treated effluent derived from 

less advanced technology is even 

more unique. 

Upstream of Wastewater Treatment Plant

Emission (nm)

300 350 400 450 500

Excitation (nm) 240

260

280

300

320

340

360

380

400

Downstream of Wastewater Treatment Plant

Emission (nm)

300 350 400 450 500

Excitation (nm) 240

260

280

300

320

340

360

380

400

Region 

1
55

Simultaneous Determination of Sulfur Isotopic Composition and Concentration in Environmental 

Samples Using a 33S/36S Double Spike Technique 

J.L. Mann, W.R. Kelly, and R.D. Vocke (839) 

Recently the National Research Council (NRC) identified several “Grand Challenges” in 

environmental research for the next generation. Included in these is “understanding 

Earth’s major biogeochemical cycles and their interaction with the global climate.” The 

biogeochemical cycling of sulfur in the atmosphere and the formation of sulfate aerosol 

particles has important consequences for global climate. The variability of sulfur isotope ratios in nature, caused by mass-dependent fractionation during biogeochemical 

processing, provides a chemical tool for tracing the various sources of sulfur aerosols 

and a useful tool for understanding the sulfur cycle. Snow and ice cores provide archives of the sources, sinks, and processing of sulfur that reflect changes in this cycle 

through time. These archives can be used to assess the current and historical changes in 

sulfur source contributions to remote regions of the Earth including polar, temperate, and tropical regions. Typical concentrations of sulfate in these regions are 25 ng/g to 100 ng/g (ppb); thus, 300 g to 4000 g of sample is required to obtain enough sample (≈ 33 μg S) for traditional isotope 

ratio mass spectrometric (IRMS) analysis. Global 

atmospheric sulfur cycling is a dynamic process that 

varies on short timescales and these large quantities of 

sample can mask seasonal changes in sulfur sources. 

Consequently, an analytical technique that allows the 

reduction of sample needed for analysis is required to 

minimize masking and increase the resolution. 

In this study we have focused on the use of multi-collector TIMS combined with a 33S/36S internal standard for simultaneous determination of sulfur isotopic composition and its concentration in low (< 1 μg) sulfate (SO4

2-) samples. The 

isotopic ratios are stated in parts-per-thousand (‰) difference from the Vienna Canyon Diablo Troilite (VCDT) isotoperatio standard, as defined by the formula δ34S (‰) = [(34S/32S)sample/(34S/32S)VCDTstd - 1] x 1000. 

The fundamental limitation to accurate and precise isotopic ratio measurements by thermal ionization is that the measured ratio differs from the true ratio in the source as a result of instrumental fractionation during vaporization of the 

sample from the filament. To address this changing ratio and to improve precision and accuracy in the 34S/32S, a wellcharacterized 33S/36S internal standard was added to the samples and was used to calculate a fractionation factor (α) that 

corrects for this changing ratio (instrumental fractionation) to give the true ratio in the source. The 32S/33S ratio that has 

also been corrected for instrumental fractionation was used to calculate the sulfur concentration. This technique has a 

significant advantage over the IRMS technique that is limited to only isotope composition measurements. 

Three International Atomic Energy Agency (IAEA) standards were used for this study. The δ34S values (reported relative to VCDT (δ34S = – 0.3 ‰) determined were – 0.38 ‰ ± 0.15 ‰ (1s), 22.65 ‰ ± 0.04 ‰ (1s), and –32.47 ‰ ± 

0.07 ‰ (1s) for IAEA-S-1, IAEA-S-2, and IAEA-S-3, respectively. Each standard showed less then 0.5 ‰ variability 

(IAEA-S-1 ≈ 0.4 ‰, IAEA-S-2 < 0.2 ‰, and IAEA-S-3 ≈ 0.2 ‰). The uncertainties are comparable to or better then 

those reported for IRMS, the typical method used for isotope ratio analysis. 

S 

CSTL researchers developed a thermal ionization 

mass spectrometric (TIMS) method that shows considerable promise as a tool for the measurement of 

low concentration sulfate samples for both the isotopic composition of sulfur and its concentration. 

δ34S (‰)

-0.8

-0.6

-0.4

-0.2

0.0

0.2

IAEA-S-1 (δ34S (‰) = -0.38 ± 0.15 1(s), n= 11) -0.8

-0.6

-0.4

-0.2

0.0

0.2

IAEA-S-1

-0.30 ± 0.16 (1s)

-0.32 ± 0.04 (1s)

-0.30 ± 0.12 (1s)

VCDT = -0.30 
56

The figure shows the NIST result for IAEA-S-1 along with those of the Institute for Reference Materials and Measurements (IRMM, Belgium), and China’s Institute of Mineral Deposits. These laboratories used a modified gas source 

(IRMS) mass spectrometer equipped with a molecular flow inlet system to determine the absolute isotope abundance 

ratios of the three standards. All labs obtained the same δ34S value for IAEA-S-1, the benchmark standard, and IAEAS-2 within the uncertainties. This does not hold true for IAEA-S-3 where our values are the same as that obtained for 

the Beijing laboratory but is significantly different then the value obtained by IRMM. This may potentially be due to 

scale contraction that is related to the gas source technique and which is eliminated by our technique. In each case our 

uncertainties are comparable or better then those obtained by the other labs. It is important to note that these measurements were made on samples ranging from 13 μg to 65 μg, 

which are smaller then the amounts used by the other laboratories (84 μg to 125 μg). Our precisions on concentration measurements range from 0.07 % to 0.23 % (% relative standard deviation), which is comparable to or an improvement over our 

present measurement capability (0.2 %) and includes a greater 

variability in the typical sample size taken for measurement. 

The availability of TIMS instruments in laboratories around the 

world will make this technique immediately available to the scientific community and can be used for any applications requiring 

highly accurate and precise measurements of sulfur such as lowsulfur fossil fuels. 

CSTL researchers are applying this technique to low sulfur concentration snow 

and ice samples from Greenland and 

Krygstan. The data obtained will allow 

the team to examine the degree of anthropogenic contribution to the sulfur cycle on 

a seasonal basis in these remote regions, 

and to assess the spatial variability of 

global anthropogenic influence. 
57

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 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 US population uses 

dietary supplements, accounting for roughly $10 billion in sales every year. 

Development of Standard Reference Materials (SRMs) for Dietary Supplements 

S.A. Wise, L.C. Sander, and K.E. Sharpless (839) 

The enactment of the Dietary Supplement Health and Education Act (DSHEA) in 1994 by the U.S. Congress has promoted growth in the nutritional supplement industry, due in part to the way in which dietary supplements are regulated. 

DSHEA provides a legal definition of dietary supplements that classifies these materials separately from food additives 

and pharmaceutical drugs. Requirements for product labeling are less stringent than for drugs, and the burden of proof for the safety of dietary supplements is placed on the Food and Drug Administration (FDA). A variety of botanical-containing products are marketed as dietary supplements, 

e.g., St. John’s wort, ginkgo, saw palmetto, and green tea. Botanical reference materials with assigned values for active and/or marker compounds are 

needed to address two primary concerns in the botanical dietary supplements community: safety and efficacy. Potential health risks may result 

from contamination (e.g., pesticides, toxic elements), 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 composition measurements for dietary supplements. 

Beginning in 2001, NIST, FDA, and NIH Office of Dietary Supplements 

(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. The goal of this collaborative 

program is to provide SRMs for eight to ten different botanical dietary 

supplements over a six-year period. Based on NIH priorities and needs, 

NIST is developing SRMs for the following dietary supplement materials: ephedra, Ginkgo biloba, saw palmetto, bitter orange, green tea, St. 

John’s wort, β-carotene and tocopherol mixtures, and multivitamin/multielement tablets. Each botanical material SRM will consist of a 

suite of matrices including plant, extract, and finished product. The first 

SRM suite for ephedra-containing dietary supplements (SRMs 3240-

3244) has been completed (see Technical Report that follows). The second SRM suite for Ginkgo biloba has been prepared and measurements of ginkgolides and flavonoids are nearing completion. Plant and extract materials for the next 

suites, saw palmetto and bitter orange, were obtained in 2004; a carrot oil, representing a natural carotene mixture, was 

also obtained. 

NIH-ODS is also collaborating with the US Department of Agriculture (USDA) to establish the Dietary 

Supplement Ingredient Database (DSID). The DSID project will report the results of a systematic survey of supplement composition, including chemical composition of ingredients with the primary focus 

on vitamin and mineral supplements. To support the DSID project, NIST and NIH-ODS have expanded 

The US 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 US. 

More than 50% of the U.S. 

population uses dietary 

supplements, accounting 

for roughly $10 billion in 

sales every year.

Bitter Orange: The dried outer peel of 

the fruit of bitter orange has been 

used medicinally, it is an appetite suppressant. 

The availability of botanical dietary supplement matrix SRMs 

with certified concentrations of 

active/marker constituents and 

contaminants and a multivitamin/multielement tablet SRM will 

provide the measurement tools 

necessary to assess the quality of 

dietary supplements. 
58

their collaboration to develop dietary supplement matrix SRMs to include a multivitamin/multielement supplement. SRM 3280 Multivitamin/Multielement Tablets is a commercial multivitamin/multielement 

tablet which will be value assigned for concentrations of 18 elements 

and 15 vitamins and carotenoids for which label claims are made. The 

candidate material for the multivitamin/multielement tablet SRM was 

received in mid-2004, and methods development and measurements are 

currently underway. Completion of the multivitamin and multielement 

SRM is scheduled for early 2006. Acquisition of the St. John’s wort 

and green tea are in progress and will be completed in early 2005. 

Development of Ephedrine Alkaloid-Based Dietary Supplement Standard Reference Materials (SRMs) 

L.C. Sander, K.E. Sharpless, J. Brown Thomas, B.J. Porter, T.A. Butler, M. Satterfield, S.E. Long, L.A. Mackey, 

K.E. Murphy, L.J. Wood, R.D. Vocke, L.L. Yu, and S.A. Wise (839) 

The FDA issued a ruling that declared dietary supplements that contain 

ephedrine alkaloids to be adulterated in December 2003. This ruling was 

based on mounting evidence of health risks associated with the use of 

ephedra, and in effect bans the use of ephedrine alkaloids (regardless of 

their botanical origin) in dietary supplements. Ephedra-containing dietary 

supplement SRMs are intended for use in method validation and as control materials for analytical methods used in the determination of ephedrine alkaloids, and should prove to be useful to support such methods and 

to demonstrate the absence of ephedrine alkaloids in ephedra-free products. 

NIST, working in collaboration with the National Institutes of Health 

Office of Dietary Supplements (NIH-ODS) and FDA, Center for Drug 

Evaluation and Research (CDER) and Center for Food Safety and Applied Nutrition (CFSAN), has recently issued a suite of Standard Reference Materials® (SRMs) that contain ephedra. Five SRMs are available: 

SRM 3240 Ephedra sinica Stapf Aerial Parts, SRM 3241 Ephedra sinica

Stapf Native Extract, SRM 3242 Ephedra sinica Stapf Commercial Extract, SRM 3243 Ephedra-Containing Solid Oral Dosage Form, and SRM 

3244 Ephedra-Containing Protein Powder. In addition, SRM 3245 

Ephedra Suite is available and contains two bottles each of the five 

ephedra-containing materials. The SRMs are certified for levels of 

ephedrine alkaloids and selected toxic trace elements (As, Cd, Hg, and 

Pb). In addition, the level of synephrine (an ephedrine-like alkaloid present in many ephedra-free dietary supplements) 

is certified in SRM 3243, and levels of caffeine are certified in SRM 3243 and SRM 3244. Information on proximates 

(i.e., moisture, solids, ash, protein, carbohydrates, fat), vitamins, amino acids, and nutrient elements is also included 

with SRM 3244. 

The SRMs are provided primarily for use in method development and as control materials to support analytical methods 

for the determination of these constituents. In the absence of a formal regulatory environment, the SRM suites will assist manufacturers of dietary supplements to characterize raw materials voluntarily and to prevent the use of materials 

that are contaminated or adulterated. In addition, the SRMs will assist 

self-assessment of consistency and quality in finished products. The 

goal of this ongoing effort is to provide tools to the dietary supplement 

industry and measurement communities that will lead to improved 

quality of dietary supplements, and ultimately reduce public health 

risks that could potentially be associated with these products.

SRMs 3240 through 3244 represent 

the first in a series of dietary supplement SRMs to be offered by NIST 

with certified values for organic constituents and selected trace elements. 

Photograph of voucher specimen representative of plants used in the 

preparation of SRM 3240 Ephedra 

sinica Stapf Aerial Parts 

NIST provides critical ephedrine alkaloid-based SRMs that 

support FDA’s goal of ensuring the safety of dietary supplements. 

The next priorities for SRM development include: β-carotene (e.g., a mixture 

of β-carotene isomers), vitamin E (e.g., 

d-α-tocopheryl acetate/d,l-α-tocopheryl 

acetate and a mixture of the tocopherol 

and tocotrienol isomers), black cohosh, 

and a number of berry materials (e.g., 

cranberries and blueberries). 
59

7. Forensics and Homeland Security 

NIST has provided DNA standards for human identification 

since 1992, and continues to refine and extend that effort. 

CSTL has 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 depends on us for 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. 

Identification of Chemical Warfare Agents Using Temperature Programmed Microsensor Arrays 

D.C. Meier, J. Evju, R.E. Cavicchi, S. Semancik (837), and Z. Boger (Israel AEC) 

Reliable and sensitive detectors for chemical warfare (CW) gases are 

needed to protect both military personnel and civilians. 

Microsensors, particularly if they respond reliably and quickly, are 

inexpensive, consume little power, and are far more practical for 

widespread use than large, complex analytical instruments. CSTL is 

developing microsensors − for fast CW agent warning − that are 

micro-electro-mechanical systems (MEMS) and 

have many of these desired features. This work 

was supported by the Department of Defense. 

The microsensors can rapidly identify three 

chemical warfare (CW) agents and a CW agent 

simulant by analyzing the electrical responses of 

an array of four microhotplate conductometric sensors with tin oxide 

and titanium oxide thin sensing films as seen in the figure. The 

measurements were done at the Edgewood Chemical and Biological 

Center (ECBC) on the CW agents sulfur mustard (HD), sarin (GB), 

tabun (GA), and chloroethyl ethyl sulfide (CEES), a sulfur mustard 

simulant. Analyte concentrations from nanomol/mol (ppb) to micromol/mol (ppm) were determined. The microsensor arrays were 

“trained” to recognized and quantitate the agents by exposing them 

to each CW agent at four different concentrations and twenty different operating temperatures. In addition, a blank response was obtained by exposing the arrays to dry air at different microsensor temperatures. All these data were used to “train” the neural network so 

that it could recognize each agent signature relative to a dry air background. The total time needed for scanning the sensor temperature, 

collecting response observations, and performing the ANN data 

analysis is about 15 seconds. Since detection time is critical in CW 

agent monitoring, a method called recursive elimination that allows 

the scan time to be decreased was investigated, while still allowing 

for accurate identification and quantification of agents. Through this 

“pruning” process the microsensor scan time can be cut by 40% to 80%. Finally, recent results show that these sensors 

can identify low concentrations of warfare agents and simulants even when the microsensors are exposed to interferences such as diesel fuel and water vapor at concentrations that are 104

 to 106 times higher than the target species. 

NIST fabricated microhotplate-based 

conductometric microsensors have 

temperature programmed operation 

and employ artificial neural network 

(ANN) signal processing methods to 

analyze data. 

Image of a microarray device with two 

TiO2 (top) and two SnO2 (bottom) sensing elements. (The color variations and 

banding reflect differences in oxide film 

thicknesses that are the result of temperature gradients that affect chemical 

vapor deposition rates.) 
60

Similar research is underway to detect other chemical 

species to demonstrate the versatility of the microsensors 

for a variety of applications including industrial process 

monitoring, and breath analysis for disease diagnosis and 

biometric identification. 

Experimentation will continue at NIST and ECBC to examine the reproducibility and long-term stability of these 

devices, and the robustness of this approach against a 

wider range of interferences. 

Advanced InkJet Printing Technology for Trace Explosives Standards 

G.J. Gillen, R.A. Fletcher, J.R. Verkouteren, M.R. Verkouteren, and G.A. Klouda (837) 

Current national priorities in homeland security have led to 

an unprecedented level of utilization of trace explosive detection systems for counter terrorism and law enforcement. 

A critical and immediate need is to develop a chemical metrology and standards infrastructure to support the widespread operational deployment of tabletop, handheld and 

portal-based trace explosive detection systems now being 

used in airports, military installations, and in law enforcement applications. 

A new piezoelectric inkjet printer system at NIST has been 

used to prepare prototype explosive Standard Reference 

Materials on a variety of substrates. Inkjet printing potentially offers a flexible, rapid, and reproducible method for 

preparation of explosive standards. Using a single standard 

solution, a large range (106

) in deposited quantity of explosive can be achieved by changing the number of drops delivered to the sample without the need for serial dilutions. 

Using four printheads allows mixtures of virtually any composition to be prepared. The NIST printer system has been 

used to prepare prototype calibration standards by drop-ondemand printing of RDX, PETN, and TNT from isobutanol 

solutions onto various substrates. The concentration of explosives delivered in individual inkjet droplets is determined 

by GC-MS analysis or by determination of droplet diameter 

using digital camera imaging with high-frequency strobe 

illumination. 

459 462 465 468 471 540 543 546 549

-0.6

-0.3

0.0

0.3

0.6

0.9

1.2

1.5

1.8

-400

-300

-200

-100

0

100

200

300

400

500

Comparison TPS, SnO2 Sensors

Derivative of Ramp/Base Ratio

Time (s)

 dry air

 26 ppb GA

 26 ppb GB

 32 ppb HD

Ramp Temperature

Derivatives of temperature 

programmed sensing data 

from SnO2 microsensors have 

been used to identify Tabun, 

Sarin, and Sulfur Mustard. 

CSTL researchers are exploring drop-ondemand inkjet printing as a low cost, flexible, 

and reproducible method for preparation of 

explosive standards over a wide range of concentrations on almost any substrate.

0

1000

2000

3000

4000

5000

6000

7000

0 50 100 150 200 250

~ng TNT

IMS Response (arb. units)

IMS response curve for TNT standard 

inkjet printed directly on collection swipe 
61

A second sample type involves printing of digitized human fingerprints using 

RDX or TNT as the printing media. To visualize the location of the fingerprint, a fluorescent dye is co-jetted with the explosives. 

Work continues to improve the quantitative delivery of solution using inkjet 

printing. The NIST printer system is being modified to use optical particle 

counting for real-time evaluation of the number of drops ejected and the individual droplet size. The next phase of the project will be to print explosives 

containing polymers to more accurately simulate actual high explosive materials. 

Determination of Traces of Fissionable Materials Using Delayed Neutron Activation Analysis 

R.M. Lindstrom, E.A. Mackey, and G.P. Lamaze (839) 

Delayed Neutron Activation Analysis is being established at NIST for the measurement of small quantities of fissionable nuclides such as 235U and 239Pu.

Since tiny traces of fissionable uranium or plutonium 

can be left behind when these materials are handled 

or transported, this analytical method can be applied 

to the analysis of “swipe” samples, an important tool 

in nuclear forensics. Using neutrons from the NIST 

research reactor, the delayed neutrons from fission in these traces can 

be used to detect and quantitate U and Pu in swipe samples. 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, matrix independent, 

and nondestructive, while being specific and sensitive. The system being built at NIST is calculated to have a detection limit for either of 

these species about 10 picograms, based on a straightforward extrapolation from published practice. The analysis time is less than 2 minutes 

per sample. 

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 cm by 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. To improve sample throughput further, the transfer system can be readily automated with computer control in the coming year. 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 also of several fission 

products. We plan to add a gamma-ray detector into the neutron moderator to exploit this signature. In addition, the 

specificity and sensitivity of this method of analysis will be put to use in certifying trace uranium in Standard Reference 

Materials. 

Inkjet printed fingerprint containing TNT and fluorescent dye 

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. 
62

Fluorescence optical micrograph of fluorescein-RDX 

particles produced by the 

VOAG

Production of Uniform Particle Standards Using the Vibrating Orifice Particle Generator and 

Quantification by Optical Particle Sensor 

R.A Fletcher, G.A Klouda, and G.J Gillen (837) 

Currently there are no standard test materials for calibrating trace explosive detection devices like Ion Mobility 

Spectrometers (IMSs) for Homeland Defense and there is 

also a need for test particles to support basic research in 

particle removal from surfaces using air jets and swipe 

technologies. The Vibrating Orifice Aerosol Generator 

(VOAG) can produce monodisperse particles from 0.5 

μm to 50 μm made from solution. Each particle contains the same amount of desired test compound that can be controlled by the solute concentration in solution. The precision and accuracy of the VOAG was determined at NIST to be 

approximately 1% using aerosol sedimentation (velocimetry) – heterodyne elastic light scattering experiments. 

The VOAG allows a wide range of compounds to be made into uniform 

particles. This is accomplished by dissolving the solute material in the 

appropriate solvent and delivering the solution by constant pressure 

through the 20 μm orifice that vibrates at 10 kHz to 100 kHz (driven by a 

piezoelectric crystal). The number of particles made per second is the 

same as the driving frequency. The particle stream is dispersed by turbulent clean air flow and then subsequently dried in aerosol form in clean 

dry air. These dry, uniform particles of known composition are collected 

on a substrate such as a filter or impacted onto a plate or surface. One 

complication is that although the number of particles made is known and 

related directly to the crystal oscillation frequency, the number delivered 

to the substrate can be 

greatly diminished due to 

particle loss during transport from generator to substrate. 

An important addition to 

the VOAG for generation 

of standard particles would be a capability to accurately quantify the 

number of particles delivered to the substrate. The detection method 

must be on-line, non-intrusive and non-destructive. One approach would 

be to use an in-line optical particle sensor (an individual particle extinction sensor) that would count each particle that 

passes through the sensor region and that is subsequently collected on the substrate. An additional benefit of this approach is that an extinction sensor can provide particle size and particle uniformity information in a rapid manner. 

The operating characteristics and verification of precise particle-making 

capabilities using a VOAG has been demonstrated. For trace explosive 

research, we have used the VOAG to produce 8 μm fluorescein particles 

containing a trace amount of RDX (mass fraction RDX = 6.7x10-5 %) as shown 

in the figure. The particles appear agglomerated due to the 

heavy loading on the filter but were single 

particles as an aerosol. These particle-filter 

collections were examined by a commercial 

trace explosives detector based on IMS and 

gave a positive alarm for RDX. 

We are developing an in-situ particle counter that will be used in-line to detect monodisperse particles as they are being 

deposited on the substrate. Several experts in optical particle sensor design have been contacted and a system has been 

designed and will be fabricated in FY2005. The detector, which will be based on an extinction or light scattering sensor, 

will permit not only counting of the deposited particles but also a measure of particle size and uniformity. Also, we 

hope to extend the capabilities of the VOAG to produce custom biodegradable polymer spheres containing known 

amounts of pharmaceuticals to support a growing program in microanalysis of biomaterials and drug delivery systems. 

CSTL researchers are developing a method to 

produce accurate and precise particle standards 

of known composition, size, and number for testing IMS instruments, trace analysis, particle testing and for pharmaceutical research. 

The ability to make trace particle 

standard test materials in a custom 

fashion for many materials is a valuable addition to our capabilities. 

Instruments that detect trace explosives and trace drug quantities can 

be tested and their performance 

quantified. 
63

Portal Filter Particle Collection Efficiency Using Monodispersed Aerosol 

G.A. Klouda, R.A. Fletcher, and G.J. Gillen (837) 

NIST has established the capability to evaluate the performance of air 

filtration systems. Although general filter specifications are usually 

available from manufacturers, many filter manufacturers know less 

about the performance of their filters for an aerosol of a particular size 

and composition. In a time of strong National Defense against terrorist 

attacks, an existing technology has emerged as one of the most effective means of detecting high-energy explosives and identifying potential perpetrators. Trace explosives detection systems are almost entirely based on ion mobility spectrometry (IMS) that relies on efficient 

microscopic-size particle sampling to concentrate trace explosives for 

subsequent desorption (heating) of these compounds into the IMS for 

detection. 

To meet the immediate need to screen people’s 

clothing for trace explosives in a noninvasive way at 

airports and government facilities, two emerging 

portal technologies were designed to utilize air jetting and convective airflow to dislodge and transport 

microscopic particles from clothing to filters for desorption into an IMS system. However, prior to this 

work no data existed on just how efficient these filters were at collecting particles of known size and 

composition. 

Filters were evaluated using airborne particles ranging in diameter 

from 0.08 μm to 8 μm at operational airflow velocities (10 m s-1). The 

filter collection efficiency was measured by generating aerosol of 

known size using two standard methods, either a Collison Nebulizer 

for aerosolizing polystyrene latex (PSL) spheres or a vibrating orifice aerosol generator (VOAG) to produce sticky liquid oleic acid aerosol of known size. The dried and neutralized aerosol flows through a filter holder, with and without 

the filter in place, and into an optical particle analyzer to determine the particle concentration. 

The filter efficiency is calculated from the concentration measured with and without the filter present. These results will likely lead to a better understanding of what size of explosive particles are 

collected, and consequently possible ways of improving the sampling and collection efficiencies. 

Future experiments will include the use of the 

VOAG to generate an aerosol of a high explosive 

and of a plastic binder, similar to a composite explosive (for example, C4) for evaluating filter efficiency and for developing a standard test surface for IMS. NIST capability of generating and counting monodispersed aerosol can be applied to virtually any filtration system. 

A CSTL research team along with 

the Transportation Security Administration and the Department 

of Homeland Security, have characterized the collection efficiency 

of the metal-fiber filter material 

used by the manufacturers of portal systems. 

The figure shows the filter collection efficiency plotted vs. particle size for different 

type aerosols. PSL variability is due to particle bounce. Collection efficiency is greater 

when filter fibers are coated with oil or for 

sticky particles, e.g., oleic acid. 

Commercial portal 

systems are based 

on airjet or convection airflow technologies 
64

Temporal temperature distribution of a metal substrate, heated 

resistively to vaporize explosive particles in the EVAP, is revealed by infra-red thermometry. Downstream, explosive vapors are recaptured on a cooled collection grid (not shown). 

NIST Trace Explosive Vapor Preconcentrator (EVAP) Test Facility 

M.R. Verkouteren and G.J. Gillen (837) 

The work described here is part of a larger effort in 

CSTL that is focused on strengthening the chemical metrology system that supports the widespread 

operational deployment of trace explosive detectors 

needed by first responders and security screeners. 

This effort is funded by the U.S. Department of 

Homeland Security (Office of Domestic Preparedness) through the NIST Office of Law Enforcement Standards. The standard test system will be used to compare existing methods and materials used in trace explosive chemical preconcentrators, and to provide comprehensive benchmarks for future improvements to preconcentrator technologies. This project is part of a larger 

effort in CSTL focused on strengthening the 

chemical metrology system that supports the 

widespread operational deployment of trace explosive detectors needed by first responders and 

security screeners. 

The major technical challenge involved the design and fabrication of a 

robust system that integrated the ability to monitor multiple aspects of 

performance, allowed flexibility of configuration, and was fairly easy 

to operate. 

Shown in the figure is a schematic of EVAP with 

peripherals that include a regulated air flow controller, hydrator, and hygrometer, through which 

air of desired humidity (<10% to 90% relative humidity) and flow rate (<1 L/min to 20 L/min) may 

be supplied. Resistive heating of the primary grid is 

performed with an operational power supply, 

where the output voltage and amperage may be 

adjusted to give the desired heating rate (1 oC/s to 

500 oC/s). The EVAP was designed so that the two 

grids could be monitored by infra-red (IR) thermometry through BaF2 (IR-transparent) windows. 

Other components include a thermoelectric (TE) 

Peltier cooling module and an interface for future 

residual gas analysis – i.e., measurements of explosive vapors in the air stream that were not condensed on the collection grid. 

Demonstration of capabilities involved determining the collection efficiency 

of the EVAP with respect to explosive type, heating rate, and airstream velocity and humidity. These four variables all proved to be significant. Under 

the operational ranges tested, RDX – but not PETN or TNT – was collected. 

Collection was enhanced at low heating rates, low air flow rates, and high 

airstream humidity levels. 

CSTL researchers improve the sensitivity of trace explosive detection at airports and other public venues by 

establishing standards for testing chemical processing 

technologies that preconcentrate the vapors of RDX, 

HMX, PETN, TNT, and other explosives. 

Stakeholders include portal/detector 

manufacturers such as GE Ion Track 

and Smiths Detection, and the Transportation Security Administration 

(TSA), which has a broad and immediate mandate to advance and deploy 

explosive detectors at airports to 

screen passengers and luggage. 

Primary grid with 

precise heating 

controls 

Regulated 

Dry Air

Hygrometer Collection grid, 

cooled by TE on 

heat sink 

IR Camera 

IR Camera 

Hydrator

RGA 
65

Results for vapor collection of RDX on 316L 

stainless steel mesh at 70 oC. Collection was 

enhanced by slow heating rate (20 o/ s), low air 

flow (0.5 m/s face velocity on collector), and 

high water vapor content of air stream (relative humidity = 80 % at 20 oC). Error bars are 

standard uncertainties based on reproducibility of replicated ion mobility spectrometry 

measurements.

0

2

4

6

8

10

12

0 20 40 60 80 10

Relative Humidity of Air Stream

Collection Yield (%)

0

2

4

6

8

10

12

0 20 40 60 80 10

Relative Humidity of Air Stream

Collection Yield (%)

0

2

4

6

8

10

12

0 20 40 60 80 10

Relative Humidity of Air Stream

Collection Yield (%)

Air Flow = 0.5 m/s

Air Flow = 1.3 m/s

Heating Rate = 400 deg/s

Morphology and chemical derivation of collector surfaces will be explored in the next year, as well as over 25 operational variables that may govern the explosive collection efficiency of vapor preconcentrators. We have designed fractional factorial experiments to explore the significance and interrelationships among these variables, and measurements 

and multivariate analysis will be performed to characterize comprehensively the EVAP performance. 

Explosives on Surfaces: A Sticky Problem 

T.J. Bruno (838) and K.E. Miller (U. of Denver) 

With heightened awareness of homeland security issues, the detection of 

explosive or energetic materials at high sensitivity and with a low error rate 

has become a pressing priority. Explosive compounds need to be detected 

on a variety of surfaces − clothing, suitcases, shoes, etc. Every surface will 

interact with the compounds and the degree of adhesion will vary, surface to 

surface. By and large, detection of these compounds relies on getting the 

molecules off the surface and into the gas phase. While there have been 

significant advances in instrumentation for both laboratory and in-the-field application, a device to be used for airport 

security will require certification by Federal authorities. Certification will depend upon a sound understanding of device performance and thus, knowing the energetic effects of surfaces, ubiquitous in all measurement scenarios, must be 

part of the certification process. 

The most fundamental measure of the interaction strength with a surface is the enthalpy, ∆H. On solids, this describes 

an adsorption process (∆HADS), while on polymers and liquids, an absorption process (∆HSOL). The vapor-phase concentration of explosive compounds is dependent on the enthalpies associated with the surface upon which the explosive 

residues have sorbed. Surprisingly, there has been very little attention given to this important parameter, primarily due 

to the experimental difficulties associated with its determination. In earlier work on sorption, we developed a technique 

to use capillary gas chromatography to measure the surface energetics of organics on soil surrogates. We have extended 

this work to energetic materials and measured the enthalpy of trinitrobenzene (TNB), trinitrotoluene (TNT), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) sorbed on a polydimethyl siloxane surface. This surface is used as a gas 

chromatographic stationary phase, and it also is used as a lubricant, release agent, coating, and as a principal component 

in silicone rubbers. The ∆HSOL for TNB, TNT, and RDX were measured to be (57.85 

± 0.2) kJ/mol, (59.48 ± 0.2) kJ/mol, and (62.36 ± 0.2) kJ/mol, respectively. We also 

determined (by measurement of Kováts retention indices) that the energy required to 

desorb an explosive is similar to that required for C14 to C16 n-alkanes suggesting 

perhaps a surrogate for instrument calibration. Moreover, we noted that the enthalpy 

of vaporization for pure TNT and TNB are higher (by approximately 20 kJ/mol) than 

the ∆HSOL values. This means that it takes less energy to “desorb” an explosive 

molecule from PDMS or “PDMS-like” material than it would to volatilize (for example by heating) a molecule from a solid particle of the pure explosive. One potential application, suggested by this discovery, would be the development of enhanced 

polymeric release surfaces for wipe testing. 

The CSLT-led team measures 

enthalpy of energetic materials 

for homeland security applications. 

Structure 

of RDX 
66

In addition to the vapor phase measurements, we have made similar measurements in the liquid (water) phase. Here, we 

have used a clay surface as a soil surrogate. These measurements are significant in that residual energetic materials in 

the environment are a serious threat, especially in live-fire military training venues. 

For example, we measured ∆HADS for RDX = (24.7 ± 0.7) kJ/mol, while 

that for benzene was measured as (18.9 ± 1.3) kJ/mol. These data indicate that while the enthalpies are not especially high, one can expect 

RDX to persist in the environment longer than benzene. 

A NIST Reference Material to Support Explosive Device Measurements 

W.A. MacCrehan and M. Bedner (839) 

To deter 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. CSTL is providing a new reference material to 

support the quality of these measurements. 

This new reference material consists of 5 g of a rifle-type 

smokeless powder. Reference values were assigned for four 

commonly determined additives, nitroglycerin, diphenylamine, 

N-nitrosodiphenylamine, and ethyl centralite. An ultrasonic 

solvent extraction method was developed to quantitatively recover these analytes. Mean values determined by micellar capillary electrophoresis and liquid chromatography were in good 

agreement. Since the LC technique provided lower uncertainty 

in the measurements, it was used for the final value assignment. 

With the development of this smokeless powder RM, forensic 

laboratories will have a reliable material for the development 

and validation of measurement methods for improvised explosives devices. The RM may also be used as a proficiency challenge sample to test operator and laboratory performance in explosives measurements as part of laboratory accreditation 

activities under American Society of Crime Laboratory Directors/Laboratory Accreditation Board (ASCLD/LAB). In 

addition, US military laboratories can use the additive reference values in the NIST RM to assure accurate measurement 

when assessing the stability of munitions. 

The smokeless powder RM 8107 was developed to help 

assure the quality of ‘low explosives’ type measurements. However, terrorist explosives’ incidents often 

involve the use of ‘high explosives’ such as TNT and 

RDX (ingredients of military and plastic explosives). In 

conjunction with NIST’s Offices of Law Enforcement 

Standards (OLES) and the Department of Homeland 

Security, we are developing a reference material for 

these high explosives. An inert solid matrix will be 

coated with commonly used military explosives. 

A retention plot showing the capacity factor of 

benzene and RDX plotted against temperature, in water as the mobile phase. This illustrates the environmental persistence of energetic materials such as RDX. 

In Aug 2004, NIST released a new reference material, RM 8107 Additives in 

Smokeless Powder, to support the quality 

of the forensic and military smokeless 

powder measurements. 

Pipe Bomb Ingredients and RM 8107 

CSTL researchers are working to develop a particulate explosives material that can be used to 

test explosives detection equipment used for airport security screening, for evaluation of suspicious packages by first responders, and in forensic laboratory post-blast investigations.
67

Candidate Material for an Explosive Residues on Soil Reference Material 

B.A. Benner, Jr. and W.A. MacCrehan (839)

Concerns have been raised by the detection of explosive compounds in 

the ground water surrounding our military munitions facilities. There is 

insufficient understanding of the environmental fate of the components 

leaching of incompletely and unexploded ordinance. An explosives-onsoil material would support the explosive residues measurements in the 

on-going remediation of these munition proving grounds. In addition, 

this material would be appropriate for use in verifying measurements 

that are part of landmine detection, forensic post-blast investigations, 

and trace explosives residue detection. 

A total of approximately 45 kg of bulk soil was collected at a military munitions proving ground. The bulk material was 

dried and a portion was sieved to particle sizes between 90 μm and 212 μm, mixed, and irradiated by a standard 60Co 

procedure to minimize microbial activity that might change the levels of analytes during storage. This processing 

yielded about 1.5 kg of suitable candidate material. The concentrations of key explosive compounds were evaluated in 

this candidate material. One-gram sub-samples were solvent extracted and analyzed by gas chromatography with negative ion chemical ionization mass spectrometric (GC/NICI-MS) detection for measurement of explosive compounds, 

including 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and 1,3,5,7-tetranitro-1,3,5,7-

tetraazacyclooctane (HMX), and related degradation products. The concentrations of the nine analytes including the 

high explosives, smokeless powder additives, and their microbial degradation products were determined to be in the 

range of ng/g to μg/g. 

Work continues to refine the GC/NICI-MS and liquid chromatography/mass spectrometric (LC/MS) methods for measurements of these explosive analytes in this natural-matrix material. Evaluation of the long-term stability of this soil 

will be an important component of the development of a reference material for these unstable analytes. We will monitor 

the material to determine whether 60Co irradiation was successful in eliminating any further microbial degradation of 

2,4,6-trinitrotoluene (TNT) that was noted in this soil prior to irradiation. 

Evaluation of Oleoresin Capsicum (Pepper Spray) Canisters for Chemical Content and Reliability 

E. White V, R.G.. Christensen (ret), K.W. Phinney, B.J. Porter, L.C. Sander (839), 

C. Presser (836), and R.Q. Thompson (Guest Researcher) 

Pepper spray is used by law enforcement officers to subdue noncooperative individuals. It is effective 85% to 90% of the time and 

its use reduces the number of injuries to officers and suspects, and 

the number of use-of-force complaints. The causes of failures have 

not been established, but may include low concentrations of the 

active ingredients, poor delivery, and variable subject response. 

The goal of this study is the determination of the identities and 

concentrations of the ingredients and the mechanical performance 

of pepper spray units to establish potential modes of failure and 

hazards in handling. 

A variety of products representing a cross section of those used by law enforcement agencies have been selected for 

study. The identities and concentrations of the active ingredients are determined by liquid chromatography/electrospray 

mass spectrometry and the identities of carriers and propellants by gas chromatography. Physical testing includes measurement of the number of 1-second bursts in a canister, a range test, a spray pattern test, drop tests, and, for the products 

that produce fogs, the measurement of the droplet size. Improved analytical methods have been developed for the determination of the active ingredients. 

An improved liquid chromatographic (LC) separation for eight naturally occurring capsaicinoids, the active ingredients 

in pepper spray, and two internal standards, N-vanillyl octanamide and decanamide, has been developed. Satisfactory 

quantitative results have been demonstrated for the measurement of the analytes in mixtures with positive ion atmospheric pressure ionization electrospray mass spectrometry (API-ES-MS) for detection. Authentic samples of the comNIST is developing an Explosive Residue on Soil Reference Material (RM) 

for environmental and forensic explosives analysis.

A program to characterize the chemical compositions and the physical 

characteristics of pepper spray products has been established in CSTL at 

the request of the NIST Office of Law 

Enforcement Standards and with 

funding from the National Institute of 

Justice. 
68

pounds have been purchased or synthesized and their purity determined. The analytical method has been tested on several naturally occurring capsaicin mixtures and on pepper sprays. 

New instrumentation has been developed to support this effort. A test chamber has been constructed for physical testing. Semi-automated devices have been fabricated and tested for determining canister spray capacity, for performing 

pattern and range tests, and for performing drop tests. Environmental chambers have been constructed for storage of 

canisters under controlled conditions. Phase Doppler interferometry instrumentation is presently being set up to measure particle sizes near the point of impact within the test chamber. 

Tests have been performed on several canisters that 

have failed in actual field use. The composition of one 

such canister, reported to have caused injuries during a 

training exercise, was characterized by API-ES-MS. 

The spray was consistent with a synthetic (rather than 

natural) product, and the total level of active constituents was comparable to sprays formulated with natural 

pepper extracts. A second canister reported to have 

failed during use was found to exhibit a weak spray 

pattern that was not centered in the direction in which 

the canister was aimed. 

Ethanol in Water Standard Reference Materials 

M.M. Schantz (839) 

The levels of blood-alcohol that determine whether an individual is considered legally impaired varies depending on the circumstances, State, and 

even the month in which the testing is occurring. As a result, practitioners 

in the field of alcohol testing have a need for reliable and stable standards at 

several concentrations. Most blood alcohol levels in driving under the influence (DUI) cases fall in the range of 0.1% to 0.3% (the average blood 

alcohol for a DUI traffic stop is 0.16 % to 0.17%). By providing SRMs 

with concentration levels set to legally relevant points, the accuracy of 

blood- and breath-alcohol testing will be improved. 

Accurate calibration of instrumentation, such as the one pictured here, is 

critical in areas of forensic testing where quantitative analysis directly affects criminal prosecutions, as is the case with the determination of ethanol 

in blood and breath. Blood- and breath-alcohol testing can be imposed on 

individuals operating private vehicles such as cars, boats, or snowmobiles, 

or operators of commercial vehicles like trucks, planes, and ships. Two new 

ethanol in water SRMs, SRM 1828b and SRM 1847, with six and three 

concentration levels, respectively, have been issued to replace the previous 

SRM 1828a, which had only four concentrations levels. 

The concentration levels in SRM 1828b, Ethanol-Water Solutions (Blood-Alcohol Testing: Six Levels), have been tailored to legally relevant points, specifically 0.02% and 0.04% for “zero tolerance” and occupational alcohol testing, 

0.08% and 0.1% for state drunk driving laws, and 0.2% and 0.3% for an average and high level for blood alcohol measurements. In addition, three concentration levels of ethanol in water (2%, 6%, and 25%) have been prepared as SRM 

1847, Ethanol-Water Solutions (Breath-Alcohol Testing: Three Levels), for use as reference solutions for breath-alcohol 

instruments. The SRMs were prepared gravimetrically, and the concentrations of ethanol in water were confirmed at NIST by using gas chromatography with flame ionization detection, the analytical method of choice for 

blood- and breath-alcohol testing in the forensic laboratory. Because some 

laboratories, particularly in California, are required to verify their primary 

standards of ethanol in water by using titrimetry, the National Metrology 

Laboratory (CSIR-NML) in Pretoria, South Africa provided measurements for each of the nine concentrations of ethanol in water using a titrimetric method, shown to be very precise and accurate. The National Analytical Reference 

CSTL is providing a reliable set of quantitative 

chemical and physical measurements on a representative set of canisters that is expected to provide a 

basis for determining the most likely causes of failures in the field and to provide a benchmark against 

which manufacturers can assess the performance of 

their products. 

The redesigned concentration 

levels in NIST Ethanol in Water 

SRMs have made them more 

useful to the forensic community 

for use as reference solutions for 

breath-alcohol instruments. 

SRM 1828b is tailored to legally 

relevant reference points. 

SRM 1847 is for calibration of 

breath-alcohol instruments 
69

Laboratory (NARL) in Sydney, Australia (another national 

metrology laboratory) also provided measurements for four 

of the solutions (0.08%, 0.1%, 0.2%, and 6%) using an exact matching isotope dilution-gas chromatographic method, 

also demonstrated to be a very precise and accurate method. 

The certified concentrations of ethanol in water in the 

SRMs are based on a combination of the gravimetry, NIST, 

CSIR-NML, and NARL measurements. The relative expanded uncertainties for the certified concentrations are less 

than 1.2% for each concentration level. 

Raman Libraries 

S.J. Choquette (839) 

Portable Raman spectrometers are currently in use by 

first responders − hazardous materials (hazmat) specialists, law enforcement officers, safety and security 

personnel − to identify unknown substances in realtime with minimal handling. Such materials include 

explosives, drugs of abuse, and bio/chemical agents. 

The issue with spectral libraries in general is that the 

origin, purity, and physical state of the compound are 

typically never specified. It is also quite infrequent 

that the instrument state of the analyzing spectrometer is adequately known. Although parameters such as spectral resolution may be listed, the calibration state of either 

axis (wavelength and intensity) are rarely given. Quality systems, which might assure the veracity of the data, are infrequently, if ever, used for spectral acquisition. Raman spectral libraries have the additional challenge in that the spectra are instrument dependent, due to the variety of laser excitation wavelengths used and nature of the measurement. 

Because Raman is an emission process, the spectra are convolved 

with the instrument response. This is quite varied from vendor to 

vendor, even given the same laser excitation wavelength. The goal 

of this work is to develop a standardized protocol for measurement 

of these compounds by Raman spectroscopy and then develop the 

protocols for transferring this reference data to commercial instrumentation. 

To this end, 14 POPs 

were chosen as a test set 

of compounds based upon input from Raman vendors and NIST’s prior experience in developing SRMs based upon these materials. GC-FID and differential scanning calorimetry were employed to verify the purity of the materials. Raman spectra were acquired on research grade instruments employing 514 nm, 785 nm and 1064 nm laser irradiation. Each instrument was 

calibrated in both x and y axes using NIST SRMs. Spectra were corrected 

for scattering and incorporated into a searchable library. Arrangements are 

in progress to measure these compounds using commercial grade systems to 

assess the efficacy of software algorithms to identify the 14 POPs utilizing 

these reference data. A collaboration has been initiated with a NIST mathematician to develop algorithms for automated Raman spectral searches. This 

is an important and necessary extension of this work to compare instruments 

of widely disparate capabilities. 

An additional solution of the ethanol in water azeotrope will be made available in FY05 

as SRM 2900. Feedback from customers 

indicated that the azeotropic mixture is 

needed in addition to those levels now available in SRMs 1828b and SRM 1847 for use 

as a starting point in the preparation of the 

customers’ working calibration solutions. 

NIST provides both the physical standards and 

the validated Raman spectral libraries to: 

• impart confidence in Raman measurements,

• provide measurement traceability to national standards, 

• improve the Raman determination efficacy, 

• ensure evidentiary acceptance of Raman 

measurements.

In 2004 a test set of compounds comprising 14 persistent organic pesticides (POPs) 

was chosen for assessment and to aid in 

the development of standardized protocols 

for inclusion of compounds into a spectral 

library. Efficient mechanism and methods 

for transferring this data to the commercial sector is ongoing.

Commercial Portable 

Raman Spectrometer 
70

NIST Support of the CDC Laboratory Response Network for Chemical Terrorism 

K.E. Murphy, M.M. Schantz, G.C. Turk, B.A. Benner, Jr., T.A. Butler, and L.J. Wood (839) 

NIST evaluated the stability of CN in frozen blood-based proficiency standards, which are used by the Centers for Disease Control and Prevention (CDC) to assess measurement capabilities of members of the Laboratory Response Network (LRN), a network of national laboratories that are equipped to rapidly test for human exposure to chemical and 

biological weapons. Cyanide (CN) has a long history of use as a chemical weapon and the need for methods and standards for measurement of CN was identified by CDC as a top 

priority. CN exposure is indicated by measuring the CN content of whole blood, however CN is very reactive and blood 

CN concentrations can decrease or increase over time depending on the method of storage. As a result, there are no commercially available CN standards to underpin the accuracy of 

blood CN measurements. CDC contracted a commercial standards supplier to produce a suite of blood-based CN standards. The standards are composed of a blank level and three 

elevated levels containing CN concentrations of nominally 

0.075 mg/kg, 0.3 mg/kg, and 1.5 mg/kg. Standards are supplied as 0.5-mL aliquots of the CN-spiked blood packaged in 

5-mL headspace vials and stored frozen at –50 °C. We have 

accurately measured the CN content of each level and have 

monitored the CN concentrations for over one year.

CSTL scientists developed a new method for the measurement of CN based on headspace gas chromatography/mass spectrometry (GC/MS) using a labeled cyanide 

internal standard (K13C15N). In addition we have completed measurement of the CN content and stability of 

CDC supplied proficiency standards. Measurements of 

five samples from each level were performed on a bimonthly basis for a period of 14 months. The CN concentration remained stable for the nominal 0.3 mg/kg and 1.5 

mg/kg levels, but increased slightly for the blank and 

nominal 0.075 mg/kg (low) level. The figure shows the 

data for the 0.075 mg/kg level, in which the measured 

CN concentration is plotted versus analysis date, and the 

solid black line shows the trend in CN concentration. In 

general, the average CN concentration for all levels was 

higher than the target nominal values. This may be partly 

due to endogenous CN in the blood stock, but may also 

be a result of the artifactual production of CN from freezing the blood. Overall, results for the three elevated levels show a variability of less than 6 % relative (1s, n = 

33). These results demonstrate a considerable improvement over data reported in the literature for the stability 

of CN-spiked blood standards and validate the viability 

of storage at –50 °C. 

An integrated public health system is vital in the event of a chemical terrorism attack. In the past two years Congress 

has appropriated $95 million for chemical terrorism preparedness. There are currently 62 public health laboratories in 

the chemical component of the LRN, over 40 of which are Level 2 designate laboratories. Level 2 laboratories are 

tasked with the analysis of CN, toxic metals, and lewisite in human samples. Inclusion in the network requires participation in a rigorous quality assurance program, which includes the analysis of proficiency standards. The data NIST has 

provided to characterize the CDC blood-based proficiency standards will help ensure that network laboratories provide 

accurate measurements in the event of a chemical terrorism incident involving CN. 

The analytical method developed in CSTL has 

been adapted by CDC for use by the Laboratory 

Response Network. 

Low Level 

0.075

0.125

0.175

0.225

06/28/03 08/17/03 10/06/03 11/25/03 01/14/04 03/04/04 04/23/04 06/12/04 08/01/04 09/20/04 11/09/04

Date Analyzed

CN (μg/g)

Plot showing the stability of CN in frozen bloodbased proficiency standards over a 14-month period.

CSTL researchers have developed a new 

method for the determination of cyanide in 

human whole blood and used the methodology 

to evaluate the stability of cyanide in frozen 

blood-based proficiency standards. 
71

Characterization of the Catalytic Domain of Class I Adenylyl Cyclase from Yersinia pestis 

S-K. Kim, S. K. Reddy, and P. Reddy (831) 

Yersinia pestis bacterium is the causative agent of bubonic 

and pneumonic plague. Because of its potential for major 

public health impact and as a bioterrorism agent, Y. pestis

has been classified as a Category A pathogen by the Center 

for Disease Control and Prevention. Adenylyl cyclase (AC) 

is one of the important virulence factors in the pathogenesis 

by bacterial pathogens such as Bacillus anthracis.

The Yersinia pestis chromosome has two genes for adenylyl 

cyclase, cyaA and cyaB. The cyaA gene encodes 850 amino 

acids (aa) Class I adenylyl cyclase (YpAC1) and the cyaB 

gene encodes 179 aa Class IV adenylyl cyclase (YpAC2). 

YpAC1 is the subject of this study. YpAC1 has discrete 

amino terminal and carboxyl terminal domains. These domains interact with each other to produce a low-activity 

form of the enzyme. 

Removal of the carboxyl terminal domain by genetic manipulation resulted 

in a free amino terminal catalytic domain with enhanced adenylyl cyclase 

activity. The activity of the amino terminal domain was found to be fourfold higher than that of the full-length 

holoenzyme. This result suggests that 

the carboxyl terminal domain is inhibitory to the catalytic function and that 

this carboxyl terminal domain regulates 

the activity of the amino terminal domain. Mutagenesis studies revealed 

important amino acid residues for catalysis in the catalytic domain. 

Mutagenesis of Asp-114 and Asp-116 

resulted in a complete loss of activity. 

This Asp-x-Asp catalytic signature is 

conserved in the infamous Bacillus 

anthracis adenylyl cyclase toxin. Adenylyl cyclases have been definitively 

shown to be virulent factors in bacterial 

pathogenesis. For these reasons, the 

adenylyl cyclase gene is an important 

target for gene knockout experiments 

and is also a useful working model to 

produce a vaccine against Y. pestis. 

Gene knockout experiments are 

planned with other Federal Laboratories having a Biosafety Safety Level 3 

capability. 

The NIST study was aimed at understanding, on a molecular basis, the connection between the fundamental genetic 

information that defines Y. pestis and the 

presumptive virulence factor adenylyl 

cyclase. This type of understanding 

could lead to a basis either for therapeutic intervention or for a vaccine for this 

biothreat agent. 

Yersinia pestis adenylyl cyclase has discrete amino terminal and carboxyl 

terminal domains. These domains interact with each other and the protein 

complex exhibits low adenylyl cyclase activity.

Removal of carboxyl terminal domain by genetic manipulation results in a free 

amino terminal catalytic domain with enhanced adenylyl cyclase activity and 

thereby produces high concentration of cAMP in the cell.

ATP cAMP

Carboxyl

terminal 

domain

ATP cAMP

ATP cAMP

Interaction of carboxyl terminal domain with sugar transport regulatory 

proteins in the cell results in a weaker interaction between the amino and 

carboxyl terminal domains resulting in a free amino terminal domain with 

enhanced adenylyl cyclase activity with concomitant high cAMP concentration.

Carboxyl 

terminal 

domain

Amino terminal Domain

Amino terminal Domain

Sugar

Transport

Proteins

Amino terminal 

Domain

The highly active adenylyl cyclase utilizes ATP, the primary source of 

energy in cells, and produces a supraphysiological concentration of 

cyclic AMP (cAMP). In anthrax disease, an elevated concentration of 

cyclic AMP inhibits the activity of the gene transcription factor NF-kB, 

a protein that would normally stimulate the production of several inflammatory factors that coordinate immune response. Cyclic AMP 

thus disables the proteins in the signaling cascade. This leads to the 

cell death of the host. 
72

Evaluation of Genetic Variation in Major US Population Groups using Human Identity Testing 

Markers 

J. M. Butler, M.C. Kline, P.M. Vallone, J.W. Redman, A.E. Decker, and M.D. Coble (831) 

A set of approximately 650 anonymous population samples from US Caucasians, African Americans, and Hispanics (self-declared ethnicities) have been 

purchased from a commercial blood bank. Over the past two years, these samples have been characterized across a variety of genetic loci used in human identity testing. Results from these samples are being used to evaluate performance 

of individual markers and various combinations of loci to enable differentiation 

of the samples. Concordance studies have also been performed with these samples between in-house multiplex polymerase chain reaction (PCR) assays and 

commercial kits to verify the absence of allelic dropout due to PCR primer binding site mutations. 

To date a total of 8 manuscripts has been published or submitted describing our 

results across these samples. Over 85,000 allele calls have been made so far on 

these samples. These population samples will likely become some of the most 

well characterized samples in the world. Decisions are being made about useful 

loci to pursue in future assays that are developed at NIST based on variation 

observed in these samples. 

These samples have also been useful in a beta-test of a 

new commercial kit for Y-chromosome short tandem 

repeat (Y-STR) amplification being released in December 

2004 by Applied Biosystems. Future plans include testing 

new loci in this set of samples.

Microfluidic Devices for Rapid DNA Analysis for Human Identification 

W.N. Vreeland, L.E. Locascio (839), M.Gaitan, J. Geist, J. Shah (EEEL), N.Y. Morgan, P. Smith, 

T. Pohida and J. Kakareka (NIH), C.W. Kan and A. E. Barron (Northwestern University) 

The National Institute of Justice estimates a backlog of a 542,700 

cases for DNA analysis as of April 2004 and current forensic crime 

labs do not have the equipment capacity to address this backlog in 

a timely manner. Forensic DNA analysis or “fingerprinting” involves the measurement of the molecular size of several fragments 

of DNA produced in a specially designed molecular-biological 

reaction. 

The NIST microfluidic device has auxiliary optics, pneumatics, and software to allow for rapid analysis of DNA “fingerprints” for human identification. The microfluidic devices are fabricated from common low-cost commercial plastics facilitating their application as single-use devices and thereby eliminating concerns of sample-to-sample cross contamination. Current device design and configuration are approaching the performance in salient figures-of-merit to current state-of-the-art equipment with the time required for analysis being reduced by nearly 90%. 

The focus of this research 

work was to examine the 

ability of commonly used 

and new genetic markers to 

differentiate between samples of US populations. 

Information collected from 

these study samples is being 

made available over the 

Internet through the NIST 

STRBase website: 

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

ch/strbase/NISTpop.htm

The tops of 95 sample tubes are shown each at a 

concentration of approximately 1 ng/μL with 

different color labels representing the various 

ethnicities. 

NIST’s development of a microfluidic device 

for forensic DNA analysis is addressing a 

major sample backlog by allowing for analysis techniques that are both faster and more 

economical, while still ensuring the data created are of the highest quality. 
73

The plastics that are used to fabricate NIST’s microfluidic devices must fulfill a variety of chemical, mechanical, and 

optical properties. In particular they must have (1) low permeability to aqueous buffers and salts, (2) low electrical 

conductivity, (3) glass-transition temperature between 110 oC and 120 oC, (4) low optical fluorescence, (5) high optical 

transparency, and (6) uniform thicknesses. Several commercial plastics were screened, and a medical-grade 

poly(methlymethacrylate) was determined to be the best match to these criteria (this is the same plastic that is used to 

fabricate disposable contact lenses for vision correction). 

The microfluidic channel must also be 

of a particular architecture and design 

(see figure for a general device layout). 

Two physical parameters of the device 

design are important, including separation channel length and sample injector 

size. The separation channel length 

determines the time required for the 

analysis as well as its analytical selectivity, while the sample injector determines the sensitivity and efficiency. In 

both cases, the desirable aspect of one 

parameter is increased at the cost of the 

other. We have design, fabricated, and 

tested a systematic assortment of microfluidic channel designs to determine the 

most appropriate physical parameters 

for the NIST forensic DNA analysis. 

The final device design had an injector 

size of 100 μm and a separation channel 

length of approximately 9 cm. Results 

from this device are below. 

Invention of a New Class of Ultra-Fast, Ultra-sensitive Mass Spectrometers for Kinetics, Reference 

Mass Spectrometry, and Homeland Defense Applications 

J. W. Hudgens (838) 

Details provided in the Measurement Science for Future Standards and Technologies section of this Report. 

4 6

18

20

22

24

26

400

380

360

340

320

300

280

260

240

220

200

190

180

160

140

120

100

90

70

80

FAM

Intensity

Time (min)

A demonstration of the NIST microfluidic DNA analyzer, showing 

the separation of a DNA “ladder” 

consisting of a variety of fragments 

of increasing size. Each peak 

represents a DNA fragment of particular size with larger fragments 

appearing later in time. The total 

analysis is performed in approximately 7 minutes. 

Top and side view of the single-channel microfluidic device 

currently being used in DNA separation experiments at NIST 

(not to scale). 
74
75

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 $1.7 trillion 

annual costs of healthcare in the US. A significant 

portion (25% to 30%) of these measurements are 

performed for non-diagnostic reasons, such as retests, error prevention, and detection. The measurement infrastructure provided by NIST supports traditional clinical markers such as the measurement of cholesterol 

and calcium in serum, and newer protein based makers such as troponin, homocysteine, and folate, as well as DNAbased standards for HER2 testing standards and Fragile X Syndrome diagnosis. Technical details on the development of 

the newer standards are provided in this chapter.

In addition, CSTL provides the clinical community with standards needed to meet regulatory requirements, both national and international. For example, during FY 2004 the European Union Directive on In-Vitro Diagnostics (IVD) 

went into effect mandating that all devices sold in the EU be traceable to “higher order” standards and methods. CSTL 

worked with the IVD industry and the international community to ensure that US devices meet the traceability requirement so that US exports to the EU would continue without disruption. As the IVD Directive went into effect increased 

demand for clinical standards has been evident; NIST noticed a dramatic increase in the sales of NIST electrolyte in 

serum SRMs. In order to expand the availability of clinical SRMs and meet the traceability requirement, CSTL continues to develop both reference methods and standards and, along with the private sector, is investigating the feasibility of 

a Clinical Reference Laboratory Network. 

Technical Procedures for a NIST-Traceable Clinical Reference Laboratory Network 

G.C. Turk, S.E. Long, and D.L. Duewer (839)

The NIST-Traceable Clinical Reference Laboratory as conceived 

seeks to provide an efficient infrastructure for supplying clinical reference measurements for IVD manufactures. Such a network could 

provide the IVD industry with reference measurements traceable to 

NIST standards in a timely and efficient manner. 

The study described here involved the measurement of calcium at 

various levels in blood serum, plasma, and urine. A subset of real 

samples of serum, plasma, and urine were measured both at NIST 

and the Mayo Clinic, using NIST calibration and validation SRMs, in 

order to establish an equivalence between NIST isotope dilution 

mass spectrometry measurements and measurements performed at 

Mayo Clinic. The study used individual patient samples that were 

split and analyzed by both NIST and the Mayo Clinic. The patient 

samples were pre-screened to cover the full range of calcium concentration for which the methods have been designed. All patient identification information was disassociated with the 

samples for this study. Further validation of the methods was provided by the analysis of NIST matrix SRMs by both 

NIST and the Mayo Clinic. These SRMs include SRM 956a (Frozen Human Serum) and SRM 2670a (Freeze Dried 

Urine). In addition, both NIST and the Mayo Clinic analyzed Candidate SRM 956b (Frozen Human Serum), which had 

not yet been certified, and for which the Ca concentration was unknown to the Mayo Clinic at the time of the analysis. 

The protocol called for the Mayo Clinic to use their normal calibration procedures, based on their in-house supply of 

calibrator solutions. They also prepared a set of NIST Calibrators by dilution of SRM 3109a (Calcium Solution). Five 

NIST calibrators were prepared at concentrations similar to those of the Mayo Calibrators. Mayo used their normal 

setup and calibration procedures and then analyzed the samples included in the study using a run order supplied by 

NIST. Each sample, including the NIST calibrators, was treated as an “unknown”, and value assigned based on the 

normal Mayo calibration procedure with their in-house set of calibrators. 

Healthcare costs amount to 

approximately 14% of the 

GDP, an estimated $1.7 trillion. By 2007, healthcare 

spending as percent of GDP is 

projected to reach over 16%. 

NIST/CSTL led a collaborative effort 

with the Mayo Clinic and DadeBehring, Inc. to test technical procedures that might be used to implement 

a NIST Traceable Clinical Reference 

Laboratory Network. 
76

The ratio of the determined values of the NIST 

calibrators relative to the prepared values using the 

NIST certified values was calculated. This value, 

and associated uncertainty, was then used to convert all of the Mayo determined values of the test 

samples (traceable to the Mayo calibrators) to values traceable to the NIST calibrators and SRM 

3109a. The conversion shift was small, but significant. The comparisons between NIST measurement results and those of the Mayo Clinic are 

illustrated graphically in the figure for each sample 

matrix. Initial results have been encouraging, and 

could potentially lead to the development of a 

network of laboratories with an expanded analyte 

and matrix coverage. 

Development of Reference Methods and Reference Materials for Clinical Diagnostic Markers 

M.J. Welch, D.M. Bunk, S. S-C. Tai, N. Dodder, B.C. Nelson, M.B. Satterfield, and L.T. Sniegoski (839) 

NIST has a long-standing effort to promote accuracy in healthrelated measurements through development of reference 

methods and SRMs. SRM 2921 Cardiac Troponin Complex 

was issued in May 2004 and has been well received by the in 

vitro diagnostic industry. The material was developed with the 

assistance of the American Association of Clinical Chemistry 

(AACC), the International Federation of Clinical Chemistry 

(IFCC), and the manufacturers of clinical cardiac Troponin I 

(cTnI) assays. Since it is a widely used marker for detecting 

that heart attacks have occurred, more reliability in the assays 

was needed. SRM 2921 is a buffer solution containing a complex of heart muscle proteins, troponins I, C, and T and certified for the concentration of troponin I. 

Certification of the concentration of cTnI in SRM 2921 was accomplished by two analytical 

methods, including amino acid analysis. Because of the inherent structural complexity of human proteins and because this complexity can have a substantial impact on immunoassay response, a thorough investigation of the chemical heterogeneity of cTnI and the other two troponin subunits, cTnT and cTnC, in SRM 2921 was performed. A commutability study is underway, involving nineteen cTnI assays currently on the market worldwide. Once accomplished, this demonstration of the commutability will put SRM 2921 in compliance with ISO 

15194 and eliminate the last hurdle for this new reference material to be acknowledged by the 

Joint Committee for Traceability in Laboratory Medicine (JCTLM) as a higher order reference 

material for use worldwide. 

A NIST-Traceable Clinical Reference Laboratory Network would become an important 

component within the clinical chemical 

measurement infrastructure, with a positive 

impact on the accuracy of such measurements and on the quality and cost effectiveness of our healthcare system. 

Serum

4

8

12

16

4 8 12 16

[Ca]NIST, mg/dL

[Ca]Mayo, mg/dL

[Ca]Mayo = -0.22 + 1.011[Ca]NIST

Plasma

4 8 12 16

[Ca]NIST, mg/dL

[Ca]Mayo = -0.13 + 1.014[Ca]NIST

Urine

0

100

200

300

400

500

600

0 100 200 300 400 500 600

[Ca]NIST, mg/dL

[Ca]Mayo, mg/L

NIST Calibrator

Calibration Line

Dimension Calibrator

Dimension

SRM

Sample

[Ca]Mayo = 1.12 + 0.978[Ca]NIST 

With implementation of the EU IVD Directive, it has become even more critical for 

NIST to develop new reference methods and 

SRMs to provide traceability for the US IVD 

industry so that this industry can maintain 

its strong position in European markets. 
77

Reference method development has been completed 

and the methods published for thyroxine (T4) and 

triiodothyronine (T3), important markers for 

evaluating thryoid function, and cortisol, an important 

hormone in metabolism. These methods are all based 

on isotope dilution liquid chromatography/mass 

spectrometry (ID LC/MS). Tandem mass spectrometry (MS/MS) is used to provide greater 

specificity in the measurements. These methods will be applied to the certification of a new hormones-in-human-serum 

SRM. This new SRM consists of two pools, one from normal adult males and one from normal, premenopausal adult 

females. Research is underway to develop new LC/MS-based reference methods for estradiol, progesterone, and testosterone, important hormones in development and reproduction. Improving the accuracy of clinical assays for hormones 

will improve diagnoses and result in earlier treatments, before serious health effects occur. This work should lead to 

capabilities for reference methods for synthetic pseudo-testosterone substances reportedly used by athletes to enhance 

performance. The new methods and the SRM will help improve accuracy of these assays and will also provide high 

order reference systems for traceability. 

Measurements have been completed for a new SRM for homocysteine (HCY) and tetrahydrofolic acid (FOL) in serum. 

This new material has three levels: (high HCY, high FOL; normal HCY and FOL; and low HCY, low FOL). This 

work is a collaborative effort between NIST and the Centers for Disease Control and Prevention (CDC), both of which 

provided measurements for certification. 

SRM 1951b Lipids in Frozen Human Serum 

was certified for total cholesterol and triglycerides (triglycerides only and total glycerides) 

this past year. This material is available, but 

additional information on HDL- and LDLcholesterol, additional heart disease risk factors, will be added 

once measurements are completed at CDC using their proposed 

reference methods for these analytes. 

The incidence of kidney disease is rising rapidly in the US. Early 

detection of kidney disease and treatment can prevent kidney failure, but early detection depends on better measurements of kidney function. Serum creatinine is the preferred measurement, but existing methods provide varying results, 

so NIST is developing a new creatinine-in-frozen-human-serum SRM to address this measurement problem. As part of 

this work, NIST has developed a new, rapid isotope dilution LC/MS method for serum creatinine to replace the tedious 

isotope dilution GC/MS method used previously at NIST. 

Renewals: Several key SRM renewals were released this year. The renewal of SRM 956b Electrolytes in Frozen Human Serum is described in detail later in this chapter. For the renewal of SRM 965a Glucose in Frozen Human Serum, 

a new low-level material was added to address measurements of patients with severe hypoglycemia. The high-level 

material was raised so that it better addresses measurements of patients with severe hyperglycemia. 

New Research: Research is continuing 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 using a proteomics approach to isolate characteristic peptides from CRP for measurement by LC/MS. An isotope-labeled peptide will be used as an internal standard for this work. 

Research has also begun to investigate the quantitative potential of Matrix-Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) mass spectrometry for biomolecules. To explore 

this potential, studies are underway on promising approaches for quantification of transferrin, an 

important iron-transporting protein in blood. Research is also underway on measuring various selenium-protein (anti-cancer agents) and iron-protein (iron-transport) combinations in blood. 

 

Tai, S.S-C., Bunk, D.M., White V, E., Welch, M.J., 

“Development (of methods for) ...Total Triiodothyronine ...” Anal. Chem, 76, 5092-5096 (2004). 

Tai, S. S-C. and Welch, M.J., “Development (of methods for) ... Total Cortisol ...”, Anal. Chem., 76, 1008-

1014 (2004). 

NIST provides New Clinical Standards for: 

Homocysteine - considered a risk factor for 

heart disease and other diseases associated 

with oxidative damage 

Tetrahydrofolic acid - a strong antioxidant, 

which is known to reduce the risk of neural 

defects in fetuses and is believed to counteract the effects of homocysteine 

Creatinine in Serum - measures kidney function for early detection of disease 
78

Reissue of Electrolytes in Human Serum SRM Now Available 

S.E. Long and K.E. Murphy (839) 

Accurate and expedient clinical assessment of patient 

health status is often critically dependent on the quality of 

clinical testing measurements, and the availability of appropriate high-quality reference materials to provide a 

traceability basis for such clinical measurements is an important requisite. Electrolytes are undoubtedly the most 

commonly measured analytes in the clinical laboratory, 

and therefore the SRM 956 series, which provides certified 

values for electrolytes in blood serum, provides an extremely important resource to the clinical measurement 

community. To support the continuing regeneration of this 

material, it is necessary to develop and maintain higherorder analytical methods, which utilize state-of-the-art 

measurement technology. 

New methods using isotope dilution inductively-coupled plasma mass spectrometry (ICP-MS) have been developed for 

the determination of lithium, total calcium, and potassium in human serum and have been used to certify SRM 956b, 

Electrolytes in Frozen Human Serum. Certification of five electrolytes, namely lithium, potassium, total calcium, sodium, and magnesium in three levels comprising SRM 956b has been completed. All of the certified values were obtained using ICP-MS, which has permitted a more efficient certification strategy. Certification measurements on the 

previous issue (956a) relied heavily on Thermal Ionization Mass Spectrometry (TIMS) and associated analytical methodology. While TIMS can provide unsurpassed accuracy and repeatability, it is an extremely slow technique. The new 

methods for calcium and potassium are based on shielded (cool) plasma ICP-MS. Significant effort was made to develop a robust method for the determination of lithium by ICP-MS. Lithium has 

not been routinely measured using this technique because of severe problems 

with instrument mass bias drift and significant inter-sample memory effects. In 

the new method these effects have been almost eliminated by the careful optimization of ICP-MS instrument conditions. 

A quadrupole ICP-MS instrument, configured with both collision cell and 

shielded plasma technology to reduce or eliminate spectral interferences has been 

installed. It is anticipated that this will provide new capabilities for the determination of clinical analytes that have been difficult to measure accurately using traditional instrumentation. 

Development of Reference Methods and SRMs for Toxic Species in Body Fluids 

S.J. Christopher, W.C. Davis, C.E. Bryan, and R.D. Day (839) 

The role of metals in health and medicine is all encompassing, with 

current research focusing on toxicology, drug delivery, degenerative 

diseases, imaging diagnostics, and development of various classes of 

metal-based drugs possessing chemotherapeutic, anti-inflammatory, 

anti-diabetic, or anti-microbial properties. Genomics and proteomics 

research will eventually lead to a better understanding of the role of 

metalloproteins in disease-proteins that may eventually serve as 

disease treatment targets. Fundamentally, all of this research is underpinned by the quality of analytical measurements 

in clinical samples like blood and urine. To address this we are developing accurate and sensitive analytical methods 

that can measure analytically difficult elements in a timely manner and producing clinical reference materials that are 

closely aligned with industry needs. 

The analytical methods currently used for electrolyte 

measurements have been 

compiled into a methods 

manual, to be published both 

as a NIST 260 Series report 

and as an on-line document 

available to external users of 

the NIST web site. 

CSTL researchers develop novel 

analytical methods for challenging 

analytes in clinical materials that 

are highly accurate and sensitive. 

Sales of NIST’s SRM 956 increased 

dramatically when the EU IVD Directive 

was implemented in December of 2003, 

and CSTL researchers developed new 

higher order analytical methods to meet 

the increased demand.
79

This work demonstrates that 

CSTL’s measurement capabilities 

can accurately benchmark the next 

generation of measurements for 

metals in clinical samples, regardless of chemical form or whether the 

samples are derived from human or 

non-human sources. 

Collision cell Inductively-coupled Plasma Mass Spectrometry 

(ICPMS) was used for the determination of interference-prone trace 

elements (As, Cd, Co, Cr, Cu, Mo, Ni, Pb, Rb, Se, Sr, V, Zn) in 

dolphin whole blood. The method is based on gravimetric solution 

handling, and an internal standard ratio-based approach to 

quantification using the method of standard additions. A mixing 

scheme that facilitated preparing and running the samples in a “dilute 

and shoot” manner was developed to avoid a time consuming, hightemperature and pressure sample decomposition. All clinical samples were prepared in a diluent consisting of several 

high purity chemicals: 1% 1-butanol + 1% ammonium hydroxide + 0.01% EDTA + 1% Triton X-100 + 1% nitric acid 

in a balance of high purity water (all by mass). The combined effects of this chemical suite serve to reduce the viscosity 

of the nebulized samples and ameliorate the differential ionization effects induced by carbon in individual analytical 

samples: 1-Butanol acts to reduce matrix effects by elevating the carbon in all matrices so that variable, sample-tosample ionization effects are minimized, especially for As and Se. Ammonium hydroxide lyses red blood cells, EDTA 

prevents the loss of metals by precipitation or absorption, and Triton X-100 prevents blockage of the ICPMS nebulizer 

and torch injector tube. The calibration scheme was streamlined to avoid the numerous sample splits typically encountered in a standard additions experiment by using the slope of a blood standard additions curve to predict the concentration in unknown blood samples successfully, with any subtle matrix effects being compensated by the internal standards. A collision cell gas was used effectively to open isotopic channels for numerous interference-prone elements, 

including As and Se, by breaking apart background species generated in the plasma. 

Another example involves the certification of methylmercury at low 

levels in SRM 966 Toxic Elements in Bovine Blood. This project 

involved development of new instrumentation and highly sensitive 

methods to address a critical problem for the CDC, which needs to 

benchmark Me-Hg measurements in clinical samples for various 

public health exposure efforts. A gas chromatograph (GC) was successfully coupled to an ICPMS via constructing a home-built heated 

transfer interface, and solid-phase microextraction (SPME) headspace sampling of microwave extracted samples was implemented to 

acquire the needed sensitivity to address this analytically challenging 

problem. Non-ideal extraction conditions can yield poor recovery or chemical transformation so several optimization 

studies were undertaken to gauge species liberation, degradation, and transformation during sample preparation and extraction, which are all circumstances presenting an obstacle 

for accurate quantification of any chemical species. Both 

methylmercury and inorganic mercury were monitored in 

SRM 966 and quantified during the method testing phase and 

the sum of the methyl and inorganic mercury species were in 

excellent agreement with certified values for total Hg. The 

method of standard additions provided the means for Me-Hg 

quantification. The chromatogram for the spiked and unspiked extracted methylmercury in SRM 966 Toxic Elements 

in Bovine Blood is shown in the figure. 

Based on this work, GC-ICPMS will be used to solve ultratrace speciation problems involving Hg and Sn chemical species, and the use and isotope dilution methods for species 

quantification will be explored. Liquid chromatography-collision cellICPMS methods will be used to certify organoarsenic compounds in a 

urine SRM. The method of double isotope dilution will be applied to a 

pharmacokinetics study. NIST staff will work closely with FDA staff that 

will assist with modeling the dynamics of orally administered MeHg in 

the blood of diamondback terrapins. Enhanced performance of the 

pharmacokinetics models will result from high-accuracy 

measurements and the ability to quantify dose and 

background levels of blood Hg among replicates independently, eliminating model noise arising from individual 

variability. 

Detection limits for Me-Hg using the 

SPME-GC-ICPMS method were 4.2 

pg/g, while identical headspace sampling and chromatographic conditions 

allowed for separation and detection of

organo-tin species at the fg/g level. 

0

20000

40000

60000

80000

100000

120000

140000

7.6 7.8 8 8.2 8.4 8.6

Time (min)

cps @ m/z 202

spiked

unspiked

The new calibration approaches developed for dolphin blood health assessments proved to be robust and applicable to similarly matched matrices of 

human and animal origin. 
80

This NIST SRM provides the fragile 

X positive control required by the 

American College of Medical Genetics Guidelines for any genetic testing. 

The Development of Two Standard Reference Materials: Heteroplasmic Mitochondrial DNA Mutation 

Detection Standard (SRM 2394) and Fragile X Human DNA Triplet Repeat Standard (SRM 2399) 

B.C. Levin, D.K. Hancock, and K.L. Richie (831) 

Human mitochondrial DNA (mtDNA) mutations are important for 

the forensics and healthcare communities. If a mutation is present in 

every mtDNA molecule, detection is routine; however, lowfrequency mutations, heteroplasmies (the existence of two nucleotides at the same site), or single nucleotide polymorphisms (SNPs), 

scattered throughout the DNA in the presence of a majority of 

mtDNA with the “Cambridge Reference Sequence” are almost impossible to detect. Therefore, NIST has developed heteroplasmic 

human mtDNA Standard Reference Material, SRM 2394, to provide 

quality control for detecting low-frequency mutations, SNPs in either 

mtDNA or in pooled nuclear DNA samples, or in heteroplasmic sites 

in mitochondrial DNA (mtDNA). SRM 2394 contains mixtures of 

two 285 base pair (bp) PCR products from two cell lines (CHR and 

GM09947A) that differ at one nucleotide position. The CHR cell 

line designated polymorphic has a T at np 6371 and the GM09947A 

cell line containing the “Cambridge Reference Sequence” has a C at 

that site. SRM 2394 is composed of 10 tubes, one tube containing 

only the DNA with the polymorphism, one tube containing the DNA 

whose sequence agrees with the “Cambridge Reference Sequence” 

and 8 tubes containing different percentages of the polymorphic/CRS mtDNA mixtures (in which the mass fractions of 

polymorphic levels are 1%, 2.5%, 5%, 10%, 20%, 30%, 40% and 50%). Before the final SRM was prepared, twelve 

laboratories including NIST participated in an Interlaboratory Evaluation (ILE) of a prototype of SRM 2394. This ILE 

was a blind study in which the investigators could use any 

mutation detection method of their choice. The methods included: automated DNA sequencing with three different 

chemistries and different sequencers; denaturing gradient gel 

electrophoresis (DGGE); the use of a designer peptide nucleic 

acid (PNA); the Luminex 100 system; the “LigAmp” procedure; and denaturing high-performance liquid chromatography. The NIST team used several of the techniques in the 

certification process, and the graph shows the NIST data acquired using the Luminex 100 system 

Fragile X Human DNA Triplet Repeat SRM 2399 to be released early in 2005: Fragile X syndrome is the most common form of inherited mental retardation and affects approximately 1/4000 

to 1/6000 males. This genetic disease has been associated with the expansion of an unstable CGG 

repeat in the FMR1 gene on the X chromosome. SRM 2399 is intended to provide clinical 

diagnostic laboratories with the quality control and quality assurance that they are correctly and 

accurately determining the number of triplet repeats in fragile X patient 

families (those individuals with normal and pre-mutation numbers of 

repeats). Late-onset of neurological symptoms has recently been shown 

in male carriers of pre-mutation alleles, a range covered by this SRM. 

SRM 2399 consists of 9 vials of polymerase chain reaction (PCR) products 

generated from DNA obtained from fragile X cell lines or patient samples. 

Each vial of PCR product contains a different number of CGG repeats. 

Expected vs. Observed Results

0 2 4 6 8 10

Percent CHR in Mixtures with GM09947A

0

10

20

30

40

50

60

Expected

Observed

In 2005, NIST will release SRM 

2394, Human Mitochondrial DNA, 

to benchmark measurements made 

for the forensic identification and for 

mitochondrial diseases diagnosis. 

Many of the procedures used in the Interlaboratory Evaluation were unable to detect the heteroplasmy if present below 

20%. This is an indication that in many 

real- life cases, low-frequency mutations 

remain undetected and that more sensitive 

mutation detection techniques are urgently needed. 

Heteroplasmy Detection of SRM 2394 Using Luminex 

100 System (NIST data) 
81

N 

T 

N 

T 

MitoChip detection of a T>C homoplasmic mutation in a lung cancer patient. Both forward 

and reverse DNA strands are sequenced on the 

MitoChip. N: normal blood, T: tumor

Forward 

Strand 

Reverse 

Strand 

Validation of Mitochondrial Changes in Cancer 

C.D. O’Connell and J.P. Jakupciak (831) 

In the past several years, homoplasmic changes have been reported in the DNA sequence of the mitochondrial genome in 

patient tumors. Changes can be identified by comparing tumor 

sequence to that of normal tissue or blood from the same individual. Lung cancer, responsible for more cancer related deaths 

than any other tumors in both men and women, remains a difficult problem for early detection, and biomarkers for this purpose 

are not currently available. 

CSTL researchers have successfully completed a pilot study 

to develop a robust, high throughput protocol to directly sequence the entire mitochondrial genome (16,568 base pairs) to 

detect these mutations. This protocol was used to validate 

reported lung cancer mutations in previously analyzed clinical 

samples. In this preliminary study, we detected mutations in 

the tumor tissue from 5 of 11 (45%) lung cancer patients. 

Further, the protocol provided 100% sequence coverage 

where DNA quality was high (19/22 mitochondrial genomes). 

This protocol demonstrated that lung cancers could be detected at increased sensitivity compared to other biomarkers 

under consideration, and was sufficiently robust to fully sequence both clinical materials with a propensity for degradation and that are large in size. The protocol was labor intensive, with the need for robotics to provide the necessary 

throughput for clinical laboratory use. To address this concern, a mitochondrial sequencing chip (MitoChip) was developed during the course of the pilot study. 

NIST scientists are evaluating the MitoChip capabilities for the detection of both homoplasmic and heteroplasmic mutations. Initial studies are promising, with mutations detected in 6/8 lung tumors with respect to the patient’s blood. Further studies are underway to address the metrics critical for reproducible, sensitive detection of tumor-specific mutations, and to validate the MitoChip in other 

body fluids useful for early tumor detection. 

This work is expected to provide a measurement base for the clinical use of resequencing (genotyping) microarrays. 

High-Throughput Bioimaging with Quantum Dots: HER2 Testing Standard 

Y. Xiao and P.E. Barker (831) 

Nanotechnology is increasingly advancing from the bench to 

commercial and medical applications. Semiconductor quantum 

dots are nanoscale binary crystals that exhibit properties intermediate between the atomic and the bulk regime. This includes high 

luminosity, stable fluorescence, and single-wavelength excitation 

for a multitude of colors. Although many applications of emerging 

nanotechnologies have been envisioned, bioimaging with luminescent quantum dots has driven this research area toward clinical application and products that cross many disciplines and platforms.

As a validation laboratory for the National Cancer Institute’s Early Detection 

Research Network (EDRN), NIST scientists are measuring the frequency of mutation and specific mutations associated 

with lung cancer. 

Manuscript submitted to the Journal of Molecular Diagnostics: 

J.P. Jakupciak, W. Wang, M.E. Markowitz, D. Ally, S. Srivastava, 

A.Maitra, P.E. Barker, D.Sidransky and C.D. O’Connell, “Mitochondrial DNA as an Early Detection Cancer Biomarker”. 

CSTL scientists demonstrate the first 

application of semiconductor quantum 

dot fluorospores to FISH testing of 

HER2 to improve measurement accuracy and reliability. 
82

Cancer detection is also evolving toward more quantitative, molecularly targeted agents that combine diagnostic and 

therapeutic features, termed “theranostics”. This class of analytes represents the vanguard of future personalized medicines. A primary example is the breast cancer diagnostic Herceptin (trastuzumab) and its target HER2, a cell-surface 

receptor gene amplified and over expressed in roughly one-third of the 180,000 new breast tumors diagnosed each year. 

Unfortunately, the tumors that overexpress HER2 are not always correctly diagnosed because of the lack of universal 

standards in clinical testing laboratories. 

Two technologies, IHC (immunohistochemistry) and FISH 

(fluorescence in situ hybridization) have been used to select 

patients whose tumors over express HER2, and who are candidates for Herceptin therapy. These methods may not always 

agree. This poses a dilemma for clinicians and for insurance 

payors. Herceptin costs up to $40,000/year/patient and carries 

side effects that include cardiotoxity in some patients. Thus, 

there is ample justification for investment in more quantitative 

analytical methods for HER2 testing. For NIST, this involves 

identification of candidate RM and SRM materials (cell lines 

and cell- free test substrates) an order of magnitude more 

complex than previous standards projects. In this case, analytes include various biological forms of HER2: the gene, the 

mRNA transcript, the protein, and the cell-surface receptor. 

The initial NIST studies demonstrated the first application of semiconductor quantum dot fluorophores to FISH testing of HER2. The 

background technology has been reviewed in context of applied genetic technology. In new work, the pH dependence of quantum dot 

fluorophores is explored in detail, and microfluidics has been recruited 

to bear on quantitation of fluorescence behavior of FISH probes detected with quantum dots. 

 

Standard Reference Material for Measuring DNA Damage Related to Disease and Aging 

H. Rodriguez, P. Jaruga, and M.M. Dizdar (831) 

Every living cell produces free radicals as part of its normal metabolism. Free 

radicals are known to result in oxidative DNA damage. This type of DNA damage has been associated with numerous age-related diseases such as cancer, 

atherosclerosis, and Parkinson’s and Alzheimer’s diseases. Free radicals can take 

over as the body’s antioxidant defense mechanisms weaken, a condition referred 

to as oxidative stress. Additionally, external sources of free radicals (such as air 

pollution, radiation, ultraviolet light and certain drugs) can tip the balance in the 

wrong direction. Oxidative DNA damage can be repaired by enzymes in normal 

cells, but for a variety of reasons the cellular repair process may fail or slow 

down, resulting in elevated levels of oxidative DNA damage that may lead or 

contribute to disease process and aging. 

The analysis techniques developed at NIST to detect oxidative damage are able to positively identify and accurately 

quantify numerous DNA base lesions caused by free radicals. Other techniques generally measure only one modificaNIST has initiated the development of a HER2 

Testing Reference Standard that will find use in 

any current or future testing platform. 

CSTL scientists pioneered 

the development of methods 

for detecting and quantifying oxidative DNA damage 

at levels of approximately 

one modified base per million DNA bases. 

Y. Xiao, P.E. Barker, “Semiconductor Nanocrystal Probes for Human 

Metaphase Chromosomes”, Nucleic Acids Research 32, (2004). 

This work has generated three lots of

candidate reference materials, and 

the beginning stages of a certification 

process for a NIST HER2 testing 

standard are underway. 
83

tion and present no structural evidence for verification; such results might be misleading and might not reflect the overall rate of DNA damage. 

A new NIST SRM (2396) is under development and will 

help scientists better measure oxidative DNA damage 

implicated in the progression of cancer and other diseases, 

as well as in the aging process. The new SRM can be used 

to calibrate methods for measuring oxidative damage in a 

DNA sample by techniques that use a combination of 

chromatography and mass spectrometry. Free radicals 

produce more than twenty different types of lesions in the 

nitrogen-containing heterocyclic compounds or "bases" of 

DNA. 

SRM 2396 contains the stable-isotope labeled analogues 

of eleven of the most-studied DNA bases modified by 

free radicals, and that of an intact nucleoside for DNA 

quantification shown in the figure. These compounds are 

used as internal standards for the measurement of analogous unlabeled lesions in DNA. Users can also calibrate 

their methods and equipment by using the components of 

this SRM. The SRM is expected to help establish measurement accuracy and consistency among different 

laboratories as well as tracea ratories as well as traceability to the NIST standard. bility to the NIST standard. 

 

Microanalytical Technologies to Support NIH Measurement Programs 

L.E. Locascio (839), M. Gaitan, N. Morgan (EEEL), P. Smith, T. Pohida, T. Phillips, E. Perruccio, and 

P. Becerra (NIH) 

NIST recently established collaborations with NIH scientists to fabricate 

microsystems based on micro-electro-mechanical systems MEMS and 

microfluidics to support and improve the measurement capabilities of 

medical researchers at the NIH laboratories. 

There are two distinct ongoing projects with different teams at NIH 

whose research involves the following: 

• Study of the immune response to Herpes Papilloma Virus (HPV) - developing chip-based microfluidic devices to be 

used in multi-analyte immunoaffinity capture and detection of proteins related to HPV in cervical secretions.

• Study of vision-related diseases involving defective neuronal differentiation or cell survival - patterned immobilization of mammalian retinal cells using microfluidic channels to guide their growth and to facilitate observation of 

their behavior under different conditions and treatments. 

Details of each of the projects are described in the following paragraphs. 

The structures of the components of SRM 

2396. Alloxan and 5-hydroxyhydantoin result 

from oxidation in aqueous solution and acidic 

treatment of dialuric acid, respectively. 

SRM 2396 is intended for use in the measurement of oxidative DNA damage by gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry 

(LC/MS), using the isotope-dilution technique 

for quantification in both cases. 

NIST and NIH researchers collaborate to enhance medical research using microfluidics and microengineering. 
84

Human Papilloma Virus: This 

work involves an epidemiological 

study of the immune response to 

the Herpes Papilloma Virus (HPV), 

for which the simultaneous isolation of multiple proteins from microliter samples of cervical secretions is required. For this purpose, 

the NIST/NIH team is 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. The system is designed so that the basic methodology can be applied to many different applications 

relevant to clinical and biological research at both institutions. 

Vision-related 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)dimethyl-siloxane (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 adhered and grew on the PEM areas preferentially as shown in the two figures. 

This team of researchers has shown that micro- and 

nanotechnology can be of great use to those involved in 

clinical research allowing for very elegant small volume 

sample manipulation and treatment as demonstrated by the early success of this work. 

DNA Detection in Microfluidic Channels

R. Zangmeister and M. Tarlov (836) 

To meet the need for real-time DNA detection in microfluidic systems, 

a unique fluorescence-based DNA microfluidic assay was developed 

that does not require labeling of sample DNA. The assay is based on the 

displacement of a short sacrificial fluorescent-tagged DNA strand by a 

longer untagged DNA sample, or target, strand as it is electrophoresed 

through a DNA-containing hydrogel plug immobilized in a microfluidic 

channel. The assay is rapid and does not require labeling of sample 

DNA. 

Microfabricated silicon/glass device 

with 4 different arms for antibody

Cervical sample 

Waste

Different 

antibody on 

each arm of 

the device

Overnight growth (approx. 18h) of retinal cells on poly(ethyleneimine). Cell 

growth is observed on the polycation 

surface areas (within the dotted black 

0.88 cm

0.88 cm

Two weeks’ cell growth on 

poly(ethyleneimine). Cells 

are covering all the polycationic area. 

0.88 cm

CSTL researchers develop a microfluidic DNA diagnostics method that 

is compatible with “lab-on-a-chip” 

platforms and can detect DNA target 

molecules in real time. 
85

This assay is designed for real-time monitoring of nucleic acid solutions by the 

electrophoresis of sample DNA through hydrogel plugs formed in a microfluidic 

channel. The main procedures of the assay are illustrated in the figure to the 

right. A DNA probe sequence designed to recognize a target DNA strand is 

chemically tethered in a hydrogel plug formed in a microfluidic channel. A 

short, fluorescently tagged indicator sequence is hybridized with a portion of the 

probe sequence (step 1), forming a stable duplex under the conditions used for 

the assay. The DNA sample to be analyzed is driven through the hydrogel by 

applying an electric field along the microchannel. If the target DNA strand is 

present, it binds to the entire probe sequence and displaces the indicator sequence (step 2). The displaced fluorescently 

tagged indicator sequence is then driven by the electric field into a second hydrogel located downstream where it is captured and detected (step 3). 

The figure displays an actual analysis of a solution containing a random 20-

mer DNA target strand. In this analysis the 20-mer strand displaces a 10-

mer indicator sequence. The displacement event signals the presence of the 

20-mer target in the analyte solution. No displacement is seen when a mismatched target strand of equal length is used. 

This approach to DNA assay explores a 

new detection method for compounds 

where fluorescent tagging in not possible, e.g., detection of toxic metals such 

as lead in blood samples. Because of 

the success of this assay, other hydrogel-based assays are being explored 

including the incorporation of catalytic 

DNA molecules that sense toxic lead 

ions in water. 

Mass Spectroscopy in Health and Environmental Science 

S.E. Stein, P. Neta, and L. Kilpatrick (838) 

The health of a cell and of an organism is reflected by the proteins that it 

contains. The recent delineation of the human genome coupled with 

radically new, sensitive methods for biomolecule detection by mass 

spectrometry has made it possible to measure a large fraction of these 

proteins, opening up a range of new, targeted methods for disease detection 

and prevention. To do this requires the ability to identify massive numbers 

of proteins, often at low concentrations and with poor separation so that any 

analysis experiment must deal with mixtures of hundreds or thousands of 

different proteins. 

The use of mass spectra for protein identification is illustrated with recent work 

from the Peptide Atlas project. In this figure, the usual reproduction of the gene 

(in black and white) is coupled with a stick diagram representing the number of 

peptides identified by their mass spectra. The colored bands are an attempt to 

show whether there are more (green) or fewer (red) peptides than expected. The 

project has cataloged 225,000 spectra for more than 26,000 proteins. The number 

of spectra and proteins analyzed has grown dramatically in the last few years, but 

little attention has been paid to the basic reproducibility of peptide mass spectra 

and to the development of effective means of establishing their accuracy. There 

has not been any significant measure of the reproducibility of the spectra from the 

same individual, the reproducibility of the spectra from different individuals, and 

the variations that are mapped to disease states. This information is required in 

order to establish reliable collections of peptide spectra for use in research and 

1. Load with indicator

(-) (+)

3. Detection

(-) (+)

2. Sample electrophoresis and displacement

(-) (+)

1. Load with indicator

(-) (+)

3. Detection

(-) (+)

2. Sample electrophoresis and displacement

(-) (+)

1. Load with indicator

(-) (+)

3. Detection

(-) (+)

2. Sample electrophoresis and displacement

(-) (+)

2.5

2.0

1.5

1.0

0.5

0.0

Time (min)

2.5

2.0

1.5

1.0

0.5

0.0

Time (min)

2.5

2.0

1.5

1.0

0.5

0.0

Time (min)

This research has been presented and published: 

• Gordon Conference on

Microfluidics 

• microTAS 2003 

• Analytical Chemistry 

(2004; 76(13); 3655-3659). 

http://www.peptideatlas.org/ 

CSTL launches the first largescale attempt to determine the 

variability of peptide spectra. 
86

clinical studies. The mass spectra for these proteins are being generated by liquid chromatography (LC), mass spectrometry (MS), and specifically by LC/MS/MS. In these experiments the proteins are chemically broken apart by specific enzymes in well-understood and predictable ways. The resulting mixtures are separated by liquid chromatography, 

the ions are produced by an electrospray apparatus, and then the molecular ions are fragmented to give spectra that are 

characteristic of the specific peptide that has been eluted. The small components, peptides, can be identified and their 

identity tied back to the genome. In this way, peptide spectra become fingerprints of the protein and can even establish 

its particular chemical state (such as post translational modifications). This mechanism has been refined by researchers 

around the world and the ease with which the experiments can be done is a major part of the explosion in information 

on the proteome. 

The first large-scale attempt to determine the variability of these peptide spectra is being done at NIST. In this project 

we are using thousands of openly available spectra along with some specific measurements made at NIST. By looking 

at the variability created by changing the apparatus and measurement conditions in well-understood ways, we are beginning to understand the best way to establish a measure of confidence in a peptide spectrum and to define new algorithms to compensate for this variability in identification of the proteins by electrospray LC/MS/MS. An example of the 

data can be seen in the figure. Here we use a very slightly modified version of the NIST MS search software to display 

and search a given protein mass spectrum produced from instruments at NIST (both in the NIST Gaithersburg labs and 

at the NIST facilities at the Hollings Marine Laboratory in Charleston, SC) with the data for the same protein from other laboratories. 

Plans are being developed to build high-quality libraries of commonly observed peptides from both directed 

measurements of selected proteins at NIST and from 

the vast and growing information available in repositories being developed in the health science community. 

This will enable a significant increase in reliability of 

protein identification as well as enable the development of refined methods for using sequence information for matching experimentally determined peptide 

spectra. While the scale of this work is very large, the 

tools developed for use with EI mass spectrometry 

here at NIST along with tools for the specific problems 

raised with mass spectra of proteins in other institutions make the effort possible. 

NIST and NIH Unveil New HIV Bioinformatics Database 

T. N. Bhat (831) and A. Wlodawer (NIH) 

AIDS is a major health concern in both the East and the West 

and in both the developed and developing countries. Structural 

data play an important role in AIDS research, however the 

ability to retrieve structural data on AIDS-related molecules is 

far from satisfactory. Drugs provide the only proven method 

for the treatment of AIDS. While there are several web resources on AIDS-related drug development, structural information on proteins that are potential targets for AIDS are scattered and data exchange between these resources is difficult. 

Structural data play a crucial role in the development of drugs 

and in understanding drug resistance as evidenced by the fact 

that a large fraction of the current drugs for the treatment of 

AIDS were developed using structure-based approaches. 

CSTL utilized existing experience with 

physical and chemical properties databases to develop a new structural 

database for AIDS research with novel 

techniques to annotate and browse. 

Publication in press: 

M.D. Prasanna, J. Vondrasek, A. Wlodawer, 

T.N. Bhat, “Application of INChI to curate, index 

and query 3-D structures”, PROTEINS: Structure, Function, and Bioinformatics (2004). 
87

Despite the wide and expanding availability and use of physical, chemical, and biochemical data collections, the ability 

to organize and retrieve structure data remains antiquated. While it is possible to query compounds whose physical 

structures are known in advance, the ability to query compounds in large, complex structural collections is unsatisfactory. To enable the bioinformatics community to have a more efficient means of accessing complex data, NIST researchers have developed a standardized techniques to annotate, index, and present structural data. This method establishes metadata, ontology, and data standards to express structures in terms of standard fragments of chemical and structural significance. 

The new NIST/NIH online database contains the structures of HIV 

protease, and compounds targeted against this enzyme. The HIV 

Structural Reference Database is a specialized resource that permits 

faster and more reliable access of standardized data related to the design and development of compounds against HIV. It also provides 

improved resources for analyzing the drug resistance for those drugs 

that are currently used to treat AIDS. The availability of such a resource to industry is anticipated to stimulate the development of new 

and better drug products. 

The HIV Standard Reference Database will annotate and distribute 

structural data on AIDS-related molecules with special emphasis to 

technological growth and drug development. The plan is to focus on 

all aspects of the data and information interoperability of structural 

data. Data standards, data uniformity, data quality, data archival, and distribution are just a few of these issues. This 

database will receive, annotate, archive, and distribute structural information about AIDS-related proteins and the compounds that inhibit them. These proteins play a crucial role in the maturation of the AIDS virus, and some of the compounds (inhibitors) are the most effective drugs thus far developed against AIDS. The database provides a crucial platform for AIDS research efforts directed toward the development of new 

drugs and treatments. This effort will also entail the expansion of the 

content of the data, the enhancement of the quality and uniformity of the 

data, the development of unique navigation and structural analysis tools, 

and the offering of comprehensive downloadable data from the resource. 

Thermodynamics of the Redox Reaction for Fatty Acid Desaturase 

V. Reipa, V.L. Vilker (831), and J. Shanklin (Brookhaven National Laboratory) 

A proper ratio of saturated to monounsaturated fatty acids 

contributes to cell membrane fluidity. Alterations in this ratio 

have been implicated in various disease states including 

cardiovascular disease, obesity, non-insulin-dependent diabetes 

mellitus, hypertension, neurological diseases, immune disorders, 

and cancer. Since desaturase enzymes play a critical role in these 

processes, they are potential targets for therapeutic intervention. 

Fatty acid biosynthesis in higher organisms has also attracted 

increased interest because of the possible use of plant oils as 

renewable sources for reduced carbon and for environmental 

cleanup. 

The mechanistic details of these reactions are just beginning to 

emerge. Thus far, the absence of thermodynamic property data 

hampers further progress in desaturase research and its applications. To address this issue, CSTL researchers have measured, for 

the first time, the redox potential E’° for the redox reaction of the 

only structurally characterized member of the desaturase family of 

enzymes. These measurements used a methodology developed in 

CSTL. By using nano-crystalline metal oxide electrodes and specThe NIST HIV Standard Reference 

Database has received about one 

million hits in the past six months. 

http://xpdb.nist.gov/hivsdb/hivsdb.html

NIST-led research team closes a critical 

gap by addressing the absence of the 

thermodynamic property data for the 

desaturase cycle. 

Recent Publication: 

V. Reipa, J. Shanklin, and V.L. Vilker, 

“Substrate binding and the presence of 

ferredoxin affect the redox properties of 

the soluble plant ∆9

-18:0-acyl carrier protein desaturase,” Chemical Communications 21 (2004) 2406. 
88

troscopic monitoring we were able to follow enzyme redox state in the presence of its cofactors. This methodology will 

be applied to elucidate the role of the different fatty acid substrates in desaturase activity. 

Potential, V(SHE)

-0.4 -0.3 -0.2 -0.1 0.0

Absorbance

0.06

0.08

0.10

0.12

b

Potential, V(SHE)

-0.4 -0.3 -0.2 -0.1 0.0

Absorbance 0.06

0.08

0.10

0.12

a' a a' a

0 SpFd 40 μmol/l SpFd

b b'

Crystal Structure of the Complex between Thrombin and the Central ‘E’ Region of Fibrin 

I. Pechik (American Red Cross and CARB), J. Madrazo, and L. Medved (American Red Cross), and 

G. L. Gilliland (831) 

A new frontier area in structural biology, X-ray crystallography, 

is providing details of the interactions of biological macromolecules. As structural genomics efforts ramp up, structures of 

many components of complex macromolecular systems will be 

determined. 

A structural investigation of the nonsubstrate interactions of thrombin with 

fibrin, which play an important role in modulating its procoagulant activity, 

was undertaken. To establish the structural basis for these interactions, inhibited thrombin in complex with a fragment, Eht, corresponding to the central 

region of fibrin, was crystallized, and the structure of the complex was determined at 3.65 Å resolution. This was a challenging effort that used X-ray 

data from twinned crystals and a phasing procedure that employed molecular 

replacement. The structure reveals a complex consisting of two thrombin 

molecules bound to opposite sides of the central part of Eht in a way that 

seems to provide proper orientation of their catalytic triads for cleavage of 

fibrinogen fibrinopeptides (see figure). 

Absorbance at 340 nm vs. the potential 

of substrate-free (○) and 18:0-ACP 

bound (●) ∆9-18:0-ACP desaturase. 

The left panel curves were recorded in 

solutions without spinach ferredoxin 

(SpFd); the right panel results were 

obtained in the presence of spinach 

ferredoxin. 

NIST and American Red Cross scientists 

have begun investigating structural 

complexes that are essential for the formation of blood clots. 

Panels A and B represent, respectively, a ribbon diagram and the

solvent accessible surface of the complex viewed along a noncrystallographic 2-fold symmetry axis perpendicular to the plane

of the page. 

Panel C represents the complex viewed along the axial projection

of the coiled coil domains. Aα, Bβ, and γ chains in panel A are in

blue, green, and red, respectively. NH2-terminal portions of the

Aα and Bβ chains and that of γ chain forming the funnel-shaped

and the γN-domains, respectively, in panels B and C have the

same color scheme as in panel A, while their remaining portions

forming the coiled coil domains are in grey. 
89

The structure was consistent with a large body of biochemical data that mapped these binding sites. This work had 

identified a large number of charge-charge interactions, 

which were evident in the structure, but the presence of a 

central region rich in hydrophobic contacts was not predicted. These findings are of significant value to future 

healthcare studies developing drugs and therapies for diseases and for blood clotting. 

Standards for Fluorescence Microarray Analyses 

G.W. Kramer, P.C. DeRose (839), and A. Gaigalas (831) 

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, singlestranded 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 100μm 

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 a 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. If artifact standards for microarray assays can be created and certified, 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. 

An initial technical workshop on fluorescence standards for microarray assays co-sponsored by Agilent Technologies 

and NIST was held in late 2002 and attended by representatives from the major array reader manufacturers. The purpose of the workshop 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 Cy3 and Cy5 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. A second technical workshop was held at NIST in the spring of 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. 

Recent Publication: 

I. Pechik, J. Madrazo, M.J. Mosesson, I. Hernandez, G.L. Gilliland, and L. Medved, “Crystal Structure of the Complex between Thrombin and the Central ‘E’ Region of Fibrin”, Proc. Natl. Acad. Sci. 

USA 101, 2718-2723 (2004). 

CSTL works with the US manufacturers of 

microarray scanners to identify specifications 

for physical artifact standards for device 

quantification. This includes: finding suitable 

fluorescent materials; identifying appropriate 

fabrication methods; and creating instrumentation for certifying the fluorescence of such 

standards. 
90

Through quarterly teleconferences, the group continues to refine parameters and to search for suitable materials. The 

working group has also developed a set of procedures and specifications for testing the suitability of candidate materials. The primary difficulty is finding materials with 

acceptable spectral characteristics that can withstand the 

high laser light intensity without photodegrading. 

The development of artifact standards for quantitative fluorescence assays on microarrays is a first step in a multirequirement process for standardizing microarray assays. 

However, it is essential that this process go forward to allow the use of microarrays in clinical settings instead of just 

research venues. Microarrays have a very promising future, 

not only in the clinical/biotech/pharmaceutical applications 

that are being developed now, but also for general chemical 

analyses. 

Reference Material 8640 for the Calibration of Flow Cytometers 

A. Gaigalas and L. Wang (831) 

The release of RM 8640 represents an important step in an 

effort to quantitate fluorescence intensity in flow cytometer 

measurements. The RM consists of six vials each 

containing a suspension of microspheres with different 

amount of immobilized fluorescein isothiocyanate (FITC). 

Each vial of RM 8640 contains a suspension of microspheres with an assigned value of “molecules of equivalent soluble fluorophore”(MESF). A significant effort was made in developing an original methodology for assigning the MESF 

values to the microspheres. The assignment is based on the comparison of fluorescence yield of the microsphere suspension with the fluorescence yield of a solution of SRM 1932. The SRM 1932 is a fluorescein solution with a certified 

concentration of fluorescein. The graph below shows the relationship between the assigned values of MESF and the 

flow cytometer response given by the channel number and is a typical calibration of a flow cytometer. The “channel 

number” refers to the digital number associated with the average height of the fluorescence pulse arising from the passage of the microsphere through the flow cytometer. This 

calibration can be used to assign MESF values to cells with 

labeled antibodies. Extension of the work to quantitation of 

multicolor flow cytometer measurements is underway. 

channel

1e+2 1e+3 1e+4 1e+5 1e+6

MESF 1e+2

1e+3

1e+4

1e+5

1e+6

NIST is collaborating with private sector firms in 

the search for candidate materials. The examination of several glass technologies with Schott Glass, 

Inc. is ongoing, and a CRADA with Evident Technologies, Inc. to study the use of nanocrystal composites has been established. 

New Reference material (RM) Released: 

RM 8640 - used for the calibration of flow cytometers that are used to detect and measure the 

number of surface receptors on living cells. The 

type and number of receptors is an indicator of 

the state and health of the cells and is of critical 

importance to health care and biothreat detection. 

CSTL researchers developed a scientific basis 

for quantitative fluorescence intensity measurements for flow cytometry. 

The six RM suspensions were mixed 

together and passed through a cytometer. The graph shows the relationship 

between the assigned values of MESF 

and the response of the flow cytometer 

given by the channel number. The 

result for the microsphere with no 

immobilized FITC is not shown. 
91

Leak 

Artifact Volume

∆V

Pressure 

Gauge

Volume, V

P0

P1

t0 t1 t2

∆

t

Region I

Volume 

V

Region II

Volume (V+

∆V)

Pressure

Time

Leak 

Artifact Volume

∆V

Pressure 

Gauge

Volume, V

P0

P1

t0 t1 t2

∆

t

Region I

Volume 

V

Region II

Volume (V+

∆V)

t0 t1 t2

∆

t

Region I

Volume 

V

Region II

Volume (V+

∆V)

Pressure

Time

9. Industrial and Analytical Instruments and Services 

Instrument manufacturers depend on 

NIST/CSTL for data, and physical and 

chemical standards data for instrument 

calibration, and 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. 

Development of a Calibration System for Refrigerant Leaks 

P.J. Abbott (836) 

In recent years, the detection of gas leaks in commercial and industrial refrigeration systems has become of paramount 

importance. Emissions of HFC (hydrofluorocarbons) have been determined to contribute to environmental problems 

such as global warming. Many countries either limit or are considering placing limits on the emission of these and other 

types of refrigerant gases. In order to ensure compliance with emissions limitations, it is necessary to quantify leaks in 

cooling systems. This requires accurate calibration of refrigerant-leak detecting equipment. 

In general, refrigerant-leak artifacts are not very accurate due to the lack of 

availability of calibration standards. Recognizing this international need, 

NIST has developed a calibration system for refrigeration leak artifacts that is 

specifically designed to account for their discharge into atmospheric pressure. 

Refrigerant leaks are detected using “sniffers” to probe suspected leaks, all 

done in situ at atmospheric pressure. This is in contrast to the more common leak detection technique that relies on 

evacuating the vessel of interest and using a mass-spectrometer-based helium leak detector to find and quantify the leak. 

Our new calibration system is based on the “Delta 

P Delta V” pressure rate-of-rise technique. This 

method measures the rate of pressure change inside 

sealed volumes in response to an inflowing source 

of gas (a leak artifact). The process is illustrated in 

the diagram. To calculate the flow rate of gas, it is 

only necessary to know the pressure rates-of-rise in 

Regions I and II, and the volume, ∆V. Because the 

flow rate is measured at atmospheric pressure, the 

temperature of the calibration system is actively 

controlled to a few millikelvins in order to minimize components contributing to the overall performance uncertainty of the leak artifact un-er test. 

In FY 2004, final system parameters were characterized, and the calibration method was tested using a leak artifact that 

had been calibrated using the NIST Primary Vacuum Flow Standard. A helium-leak artifact having 

a flow rate of approximately 4x10-11 moles per second at 23 oC was used, and the results obtained 

with the new calibration system were consistently within 2% of this value. The uncertainty budget 

for the calibration system is approximately 5%. 

Future testing at atmospheric pressure with a refrigerant leak will be done in conjunction with the 

Italian and German National Metrology Institutes (Istituto di Metrologia "Gustavo Colonnetti" and 

Physikalisch-Technische Bundesanstalt, respectively), which are concurrently developing systems 

based on different measurement processes for calibration of refrigerant leaks. This work will lead to 

a NIST calibration service for refrigerant leaks. 

CSTL research paves the way 

for a NIST calibration service 

for refrigerant leaks. 

The US is the largest producer of 

industrial and analytical instruments in the world – it is highly 

competitive, globalized and technologically advanced. Many larger US 

companies derive a significant portion of their revenues from international sales and have manufacturing

operations throughout the world. 
92

Primary Acoustic Thermometry 

G.F. Strouse, D. C. Ripple, M.R. Moldover (836), and L. Pitre (BNM/INM, France) 

The most accurate determinations of thermodynamic temperature above 700 K use relative radiance measurements 

referenced to a black body. When the black body is operated near 700 K its thermodynamic temperature can be 

determined using constant volume gas thermometry 

(CVGT). Unfortunately, two CVGT measurements made 

at NIST differ from each other for reasons that are not well 

understood. The difference has led to an estimated uncertainty of 13 mK for temperatures near 700 K, and 50 mK 

for temperatures near the gold point (1337.33 K). Similar 

inconsistencies occurred below 273.16 K, reaching 

±0.005% at 90 K and below. 

In response, NIST developed spherical and quasi-spherical, noble-gas-filled cavities that function as acoustic thermometers. We measured the frequencies of the cavities’ acoustic and microwave modes while enclosed in a high-performance 

thermostat. From the obtained data we determine the speed of sound in the noble gas, which in turn, revealed the thermodynamic temperature, T. Simultaneously, we measured the cavity-wall temperature with standard platinum resistance thermometers calibrated on the International Temperature Scale (ITS-90), thus allowing the determination of the 

deviation between the ITS-90 and the thermodynamic temperatures (T-T90). 

Microwave and acoustic data were acquired in the interval from 85 K to 505 K using two resonators (one copper and 

one steel) and two gases (helium and argon). In the limited region of overlap, from 217 K to 273 K, the results agreed 

within combined uncertainty, and the determination of (T-T90) had a standard uncertainty of 0.6 mK. The figure displays 

these data along with acoustic thermometry data from other laboratories and data that were used to establish the ITS-90. 

The NIST approach to acoustic thermometry has been 

adopted by independent groups in England and Italy, 

and a NIST Guest Researcher from BNM/INM is expected to continue acoustic thermometry below 77 K 

when he returns to France in 2005. 

We also have indications that acoustic thermometry 

might work in the range from 3 K to 24.6 K where 

NIST currently supports the ITS-90 using an interpolating gas thermometer as a primary standard. In the coming year, we shall investigate the feasibility of using an 

acoustic scale realization method, which could yield an 

easier to use and more accurate instrument. Using a 

rebuilt version of the high-temperature (steel) acoustic 

thermometer this work will be extended to 700 K and 

higher. 

NIST has developed spherical and quasispherical, noble-gas-filled cavities that 

function as acoustic thermometers. For 

acoustic thermometry, the measured 

quantities are frequencies and temperatures. 

For constant volume gas thermometry the 

measured quantities are pressures and 

temperatures; thus, the two approaches to 

thermometry have different systematic 

effects. 

Fractional deviations from the ITS-90 in ppm (106). 

The colored symbols represent acoustic thermometry; the black symbols represent gas and radiation 

thermometry used to develop the ITS-90. 

These experiments have resolved the longstanding discrepancy in NIST’s CVGT results 

and are expected be incorporated into a new 

temperature scale reducing the errors in the 

ITS-90 by a factor of 5 to 8. 
93

Atomic Standard of Pressure 

M.R. Moldover, J.W. Schmidt (836), E.F. May (Univ. of New South Wales, Australia), and Y. Wang (EEEL) 

The primary pressure standard at NIST, below 300 kPa, is a 3 meter 

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 using 

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. The extrapolation cannot 

be checked with existing technologies; thus, it is not fully trusted. 

Furthermore, piston-cylinder sets exhibit a poorly-understood species and 

gas flow dependencies. 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. 

The figure is a sketch of an experiment that compared two techniques for 

measuring ε(p,T), the dielectric constants of helium and argon in the 

ranges 0.1 MPa to 7 MPa and 0 °C to 50 °C. One technique uses crosscapacitors and a capacitance bridge. As sketched in Figure 1, the cross 

capacitor has four electrodes. In contrast, most capacitors have two electrodes. The extra electrodes compensate for surface contamination (oxides, adsorbed water, or films of oil). The symmetry of the cross capacitor 

makes it insensitive to microscopic displacements of its electrodes. The 

second technique for determining ε(p,T) measures the microwave resonance frequencies of gas-filled quasi-spherical cavity, i.e., a cavity with a 

shape that intentionally differs from a perfect sphere. (In the figure, quadrants of a perfect sphere have been cut out of the cavity and the asphericity is exaggerated.) The microwave resonances of a spherical cavity occur in overlapping multiplets; the resonances of a quasi-spherical cavity 

are separated into easily measured, non-overlapping components. 

The graph shows recent test results. We measured ε(p,50°C) of helium and argon. The center panel of the graph shows 

the differences between ε(p,50°C) measured using a cross capacitor and ε(p,50°C) measured using a quasi-spherical 

cavity. The differences have a standard deviation (S.D.) of only 8×10−8

. This standard deviation is a factor of 6 smaller 

than that of our previous report two years ago. 

The data in the graph are limited by imperfections of the 

capacitance bridge, which is the 

best one commercially available. We asked NIST’s Small 

Business Innovative Research 

Program (SBIR) to seek a 

greatly improved capacitance 

bridge. One vendor demonstrated that factor-of-10 improvements are feasible and 

won an SBIR Phase II contract 

to make a prototype bridge having such improved performance.

Today, measurements of ε(p,T) made with a quasi-spherical cavity 

have 10 times the resolution of measurements made with the 

existing capacitance bridge. However, the cavity is not yet an 

accurate realization of the pressure standard because we do not 

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. 

Comparison experiment. The dielectric constant of either helium or argon 

is measured simultaneously with a 

cross capacitor (left) and with a quasispherical cavity (right). 
94

know the elastic constant kT that determines how 

the cavity shrinks under applied pressure. The 

ultrasonic resonance technique used to measure kT

of solids gave poor results when we applied it to 

the copper cavity. We shall overcome this problem 

by constructing new microwave cavities out of 

copper-plated hard metals. 

Progress Toward Realizing Pressure with Dimensionally-Based Piston Gauges 

J.W. Schmidt, A.P. Miiller, W.J. Bowers, and D.A. Olson (836) 

An accurate determination of pressure has many 

important applications, including altitude determination of aircraft, measurement of barometric pressure, and monitoring and control of 

pressure in manufacturing processes. The pressure standard for NIST in the atmospheric range 

is presently a mercury manometer. Piston/cylinder assemblies (i.e., piston gauges) are 

an alternative means for establishing pressure; 

through use of a variety of piston diameters and 

mass loads they can operate from the atmospheric range to several hundred MPa (tens of 

thousands of psi). Dimensional measurements 

and flow models allow a direct determination of 

the effective area of a piston gauge, and therefore the pressure it can generate, without reference to another pressure artifact for its calibration. The ability to measure the dimensions of 

the piston/cylinder assemblies that are extremely round and straight have allowed us to 

contemplate piston gauge pressure standards 

whose uncertainties approach the best manometers. 

At NIST an additional independent realization 

of the pressure unit provides a means to check 

the operation of our primary mercury manometers and a primary standards having considerably greater operating range 

For the last few years, NIST has been studying two 35 mm diameter piston gauges as potential primary standards, designated as PG 38 and PG 39. The artifacts have been used as check standards for 

the NIST Ultrasonic Interferometer Manometer (UIM) for the last decade. Those comparative measurements have shown PG 38 and PG 39 to be stable within a few parts in 106

. This year, the piston 

and cylinder of both PG 38 and PG 39 were accurately dimensioned by Physikalisch Technische 

Bundesanstalt (PTB) and analyzed using a model of normal and shear forces on the base and flanks of the piston. PG 39 

was also dimensioned by PTB in 1999, and these two sets of measurements have been used to evaluate the dimensional 

stability of the artifact and the repeatability of the dimensioning method. 

This research will revolutionize the realization of pressure standards. Since gas-filled, quasi-spherical cavities are excellent acoustic

thermometers we can determine the imperfections of the internationally accepted temperature scale ITS−90. We can also measure 

more accurately the primary constituents in natural gas and these 

data can be used for state-of-the-art metering technologies. 

Realizing pressure by two independent 

techniques provides independent 

methods for realization of primary 

pressure standards and more credence 

to the uncertainties claimed by each. 

Comparison of effective area of PG39 vs Pressure in kPa 

from NIST UIM, 1999 dimensional data, and 2004 dimensional data, showing agreement to 1 in 106

. 

A mercury manometer is NIST’s pressure standard in 

the atmospheric range. This project seeks to use piston 

gauges as an alternative means for establishing pressure 

without reference to another pressure artifact. 

PG39 vs 360 kPa UIM 

Nitrogen, ABSOLUTE Mode

1007.943

1007.945

1007.947

1007.949

1007.951

1007.953

1007.955

1007.957

1007.959

1007.961

1007.963

0 50 100 150 200 250 300 350

Pressure / kPa

+ Dimensional 1999

 + Dimensional 2004

2004

2004 Linear Fit

1 part in 106

Lambda = (6.31 ± 3.0) x 10–12 Pa-1
95

The dimensional measurements indicate all four pieces are 

round to within ±30 nm and straight to within ±100 over a 

substantial fraction of their heights. Using the 2004 data, the 

model indicates an expanded relative uncertainty on the 

effective area of 4.4x10-6 for PG39. Operational effects, such 

as force determination under piston rotation, gas species, or 

gas mode, may increase the uncertainty when the artifact is 

used to generate pressure. The difference in the relative 

effective area between the 1999 and 2004 dimensional data 

for PG39 is less than 1x10-6, and the difference between the 

2004 dimensional data and a 2004 pressure comparison with 

the UIM is also about 1x10-6 also about 1x10 . -6. 

The NIST Hybrid Humidity Generator 

G.E. Scace, C.W. Meyer, W.W. Miller, J. Hodges, and D.C. Ripple (836) 

Gas streams of well-characterized humidity are generated for 

calibration of customer’s hygrometers. The NIST Hybrid 

Generator generates humidity using two distinct physical 

principles. First, because the vapor pressure of water is well 

established as a function of temperature, a gas stream can be 

saturated at a known concentration of water vapor by passing 

the gas through a saturator, which is a serpentine passage 

maintained at a known, uniform temperature that contains a 

layer of water on its lower surface. Second, for the creation 

of low moisture levels (down to 3 parts in 106

 mole fraction), 

the gas exiting the saturator can be reduced in water concentration by diluting the gas stream with dry gas. Present-day 

laminar-flow elements enable very accurate measurements of 

gas flow, resulting in low uncertainties of the water concentration of the diluted output stream. The use of a dilution 

scheme is very fast and efficient compared to operation of a 

saturator at cryogenic temperatures. Furthermore, a commercial pre-saturator initially brings the input gas to almost the 

desired moisture level, reducing the heat and mass transfer 

load of the saturator itself and allowing operation at high gas 

flow rates and high water concentrations. 

In FY04, the pre-saturator, heat exchanger, saturator assembly, and the dilution system were completed. The heat exchanger and saturator, core elements of the generator, are 

shown in photograph. To facilitate efficient operation, the 

new generator is designed to allow full automation of the presaturator, dilution system, and saturator baths. We have also 

designed the system to support closed-loop control algorithms, in which the performance of the saturator is continuously monitored and the pre-saturator adjusted to give minimum uncertainties in the generated humidity. 

In the coming year, we will compare PG 38 

against PG 39 to further verify the modeling 

technique, and propagate characterization of 

the artifacts to other piston gauge standards by 

direct calibration. This will revise the NIST 

pressure scale for gases up to 17 MPa, and provide lower uncertainties for our customers. 

The NIST Hybrid Humidity Generator 

has been designed to extend the humidity range provided by NIST, increase 

operational safety and efficiency, decrease calibration uncertainties, and 

eliminate the need for expensive cryogenic cooling. 

The Hybrid Humidity Generator is nearly completed, its operation refined and automated, 

and its uncertainty documented. Use of the generator in regular calibration service is anticipated at the end of FY05. 
96

Testing of a Nanogate Device as a Variable Flow Leak Element 

P.J. Abbott, A. Lee (836), and James White (MIT) 

Many critical industrial processes rely on generating and delivering accurate and precise flows of gas. Examples include 

leak testing of nuclear containment vessels, gas delivery in 

semiconductor processing, quantifying the emission of ozonedepleting chlorofluorocarbons, food processing and packaging, and testing of medical implants such as pacemakers. The 

gas flows required for these applications span a very broad 

range, from as low as 10-14 moles per second to greater than 

10-6 moles per second. Several types of measurement instruments are currently used to cover this range including mass 

flow controllers, metal capillary leak elements, and permeation leak elements. Recently, a variable flow leak artifact using micro-electro-mechanical systems (MEMS) technology 

called the Nanogate was developed at the Massachusetts Institute of Technology (MIT) and holds excellent potential for 

covering this range with a single device. 

The Nanogate has the potential for generating gas flows over the entire range mentioned above, for many gases of interest, and offers the advantage of precise flow-rate control due to its ability to adjust its opening on a sub-nanometer scale 

using a picomotor actuator. The Nanogate is a micro-mechanical device that is designed to control a nanometer-sized 

gap. Precise control of the gate opening is accomplished by deflecting a cantilevered plate that is anchored by a torsion 

spring, as shown in the diagram. 

To characterize operational performance of the 

Nanogate device, MIT researchers needed a way 

of generating accurately known flows of gas. 

CSTL staff members of the Pressure and Vacuum 

Group maintain low gas-flow standards over the 

range of 10-12 mol/s to 10-6 mol/s and can generate 

lower flows using a flow division technique. Initial testing of the Nanogate device with helium gas 

has agreed well with theory. A roughly parabolic 

dependence between helium flowrate and applied 

picomotor opening was obtained, and minimum 

leak rate changes on the order of 10-13 mol/s were 

obtained for 51.7kPa of helium at the inlet. Additional testing is planned with other gases over the 

complete range of the NIST low gas-flow standards. Given the fine resolution of the Nanogate opening, a minimum 

change in flow rate per applied pulse should be below 10-14 moles per second. This may make the Nanogate the most 

precisely controlled gas delivery valve in the world. 

 

Gas Standards Based on Optical Spectroscopies 

J.T. Hodges, P.M. Chu (836), and R. Ciurylo (University of Nicolas Copernicus, Poland) 

This program involves the realization and dissemination of 

primary gas measurement standards based upon quantitative 

absorption spectroscopy. Precise spectroscopic measurements are used in conjunction with low-uncertainty methods 

of sample preparation. This approach yields absolute measurements of absorption line intensities for a variety of lowrelative molecular mass target analytes such as CH4, H2O, 

NOx, O3 and NH3. 

New applications for gas standards demand 

high-precision measurements of relatively 

low concentrations for reactive and nonreactive systems motivates NIST to development of next-generation standards linked 

to intrinsic molecular properties. 

CSTL pressure and vacuum experts 

characterize flow rate characteristics of 

the MIT developed Nanogate. 

Leak Rate vs. Nanogate Opening

0.0E+00

5.0E-10

1.0E-09

1.5E-09

2.0E-09

2.5E-09

3.0E-09

3.5E-09

0 0.02 0.04 0.06 0.08 0.1

Nanogate Opening (Arbitrary Units)

Leak Rate (moles/s)
97

The goal of this program is to shift the principal realization of 

traceable gas measurement from one based on consumable 

artifacts to one based on intrinsic standards. Advances in spectroscopic measurements finally make robust intensity measurements viable for quantitative gas metrology of certain species with an accuracy rivaling and potentially surpassing traditional measurements using artifact standards, particularly for 

reactive gases. 

This novel variant of cavity ring-down spectroscopy (CRDS) 

yields absorption spectra in which the frequency axis and absorption coefficients are directly linked to high-precision frequency counting measurements and measurements of cavity 

photon decay times, respectively. We demonstrated line intensity measurements of H2O with a relative precision better than 

0.3% at H2O mole fractions of 10-5. Automation of the spectrometer was also accomplished this year. The automation 

method required the development and integration of servo systems to tune the frequency-stabilized reference laser, to frequency-lock the ring-down spectrometer to the reference laser, 

and to lock the probe laser frequency to the ring-down spectrometer, respectively. 

Measurements of weak overtone spectra of H2O made with 

the ring-down spectrometer, shown in the figures, demonstrate the sensitivity and spectral resolution of the instrument. 

As an alternative approach to existing primary and secondary 

gravimetric gas standards, the proposed technique will extend 

NIST capabilities in gas standards to lower concentrations 

than are anticipated to be achievable using current methods 

and to reactive species that are unsuitable for long-term storage in cylinders. Customers include users of NIST standard 

gas and users of molecular spectroscopy line intensity data. 

This work is relevant to emissions of toxic industrial compounds and emissions from ground and air transportation 

systems, air pollution monitoring, metrology of high-purity 

gases used in manufacturing processes, atmospheric science, 

defense, health-care diagnostics, and homeland security. 

We plan to use CRDS to measure spectral line shapes and line intensities of H2O and CH4, and we will pursue applications of CRDS to reactive gases. Comparisons to NIST primary thermodynamic and gravimetric gas standards will be 

made to maintain links with existing standards. To address measurement challenges of specific gas mixtures for which 

component species have overlapping absorption spectra, fourier-transform spectroscopy will be used to identify optimal 

wavelength regions where spectral interferences are minimized. 

H2O survey spectrum measured using frequency-locked cavity ring-down spectrometer.

Advantages to NIST customers include: lower 

uncertainties, coverage of new species, potentially lower cost for standard mixtures, more 

flexibility in terms of dilution gases, and the 

availability of low-uncertainty molecular property data applicable to spectroscopic measurements of gas concentration. 

High-resolution scan of ro-vibrational H2O 

transition obtained with frequency-locked 

CRDS technique. 

CSTL researchers developed a new approach to 

cavity ring-down spectroscopy using continuous- wave lasers that are frequency-locked to 

frequency-stabilized ring-down cavities. 
98

Characterization of the New Thermal Neutron Prompt Gamma Ray Activation Analysis Instrument 

E.A. Mackey, R.M. Lindstrom, G.P. Lamaze (839), H. Chen-Mayer (PL), P. J. Liposky (MSEL), and D.L. 

Anderson (FDA) 

A new thermal neutron prompt γ-ray activation analysis (PGAA) 

instrument was designed to provide greater sensitivities, better detection limits, and much lower background radiation. Experiments were 

performed to measure the improvements in element sensitivities and 

background count rates. 

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. Capture 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 when compared with the original instrument. The improved detection system, 

positioned closer to the sample, increased element sensitivity by 5% 

to 50%. 

During the past year work was completed on the 

determination of 0.1% N in oil materials which 

would not have been possible without the previous instrument. We have also completed a comprehensive uncertainty assessment for this method 

and this instrument. 

Effects of Backscattered Electrons on the Analysis Area in Scanning Auger Microscopy 

C.J. Powell (837) and A. Jablonski (Institute of Physical Chemistry, Warsaw, Poland) 

Auger-electron spectroscopy (AES) is a commonly used technique 

of surface analysis. For example, it is used in the semiconductor 

industry to identify unwanted particulates and other defects on wafer 

surfaces that are detected after various processing steps. In this and 

similar applications, a focused electron beam (with a width of about 

10 nm) is directed onto the feature of interest, and a measurement 

made of the surface composition. Analysts typically assume that the 

detected Auger-electron signal originates from an area defined by 

the width of the incident beam. The analysis area, however, is genCSTL’s new PGAA instrument has 

greatly reduced limits of detection 

when compared with those of the 

original instrument due to reduced 

gamma-ray backgrounds and increased element sensitivities. 

The new PGAA instrument is safer to assemble for use and 

provides greater sensitivities, better detection limits, and 

much lower background radiation than the original instrument. Analyses of SRMs and other materials will benefit 

from these improvements. Additional work is in progress to 

prepare a suite of standards for the elements of interest for a 

broad range of neutron absorption and scattering matrices. 

The CSTL-led research team seeks to 

provide reference data, models, and reference procedures to enhance the accuracy and efficiency of surface analyses 

made by Auger-electron spectroscopy 

(AES) and X-ray photoelectron spectroscopy (XPS), thereby improving the 

traceability of chemical measurements. 
99

erally much larger due to the detection of Auger electrons resulting from inner-shell ionizations induced by multiply 

scattered primary electrons in the vicinity of the sample surface (i.e., by so-called backscattered electrons). 

The research team utilized a simple analytical model to estimate the effects of backscattered electrons on the analysis 

area in scanning Auger microscopy (SAM). For normally incident electrons, the radius ra of the analysis area was calculated corresponding to detection of 80%, 90%, and 95% of the total Auger-electron signal as a function of two sample 

parameters, the backscattering factor R and a Gaussian parameter σb describing the radial distribution of backscattered 

electrons. For a reasonable range of these parameters, ra depended linearly on σb and to a lesser extent on R. Values of 

ra can be appreciably larger, by up to a factor of 100, than the typical widths of the incident beam in modern SAM instruments. 

As an example, the figure shows the ratio of the Auger signal within an area of radius ra to the maximum Auger signal 

(for an infinite radius) for an illustrative case in which σb = 200 nm and R = 1.5 (typical values for a primary-beam energy of 20 keV). In this example, two-thirds of the total Auger signal originated from ionizations by the primary beam 

and was emitted from an area of radius 30 nm. The remaining one-third of the total signal originated from ionizations 

by backscattered electrons and was emitted from a much larger area, with 90% from an area of radius about 310 nm, 

95% from an area of radius of about 390 nm, and 99% from an area of radius of about 530 nm. The parameter σb can be 

up to about 1 μm for some materials at 

a primary energy of 20 keV, and ra can 

then be up to about 2 μm (for detection 

of 95% of the total Auger intensity). 

These simulations confirmed the general trends found in the first phase. 

Values of ra (for detection of 95% of 

the total Auger intensity) ranged from 

30 nm (Au substrate, primary energy 

of 5 keV) to 680 nm (Si substrate, primary energy of 10 keV). 

Most "point" analyses by AES have been previously based on 

the implicit assumption that the entire detected signal originated 

from the feature of interest. When Auger analyses are required 

of fine particles on surfaces or of inclusions in matrices, it will 

generally be necessary to reduce the beam energy to ensure that 

most of the signal comes from the feature rather than from regions far from the feature. As a result, a tradeoff may have to be 

made between spatial resolution, analytical sensitivity, and validity of the analysis. 

Monte Carlo simulations were 

made to investigate the radial 

distributions of Auger electrons from a thin copper film 

on substrates of silicon and 

gold for normally incident 

primary electrons with energies of 5 keV and 10 keV. 

0

0.2

0.4

0.6

0.8

1

0 100 200 300 400 500 600 700

Plot of I/Imax, (y axis) the ratio of 

the Auger intensity from a circular area of radius ra to the total 

Auger intensity from a circular 

area of infinite radius, as a function of ra in nm (x axis). 

A new NIST database for the Simulation of 

Electron Spectra for Surface Analysis 

(SESSA) of multilayered thin films by AES 

and XPS will be released in FY 2005. We 

plan to extend the capabilities of this database in the future to allow simulations of 

AES spectra for fine particles on surfaces 

and inclusions within samples. 
100

Uncertainties of Industrial Thermometers 

K.M. Garrity, D.C. Ripple, G.F. Strouse, and C.D. Vaughn (836)

In spite of the maturity of commonly used industrial 

thermometers, including thermocouples, resistance thermometers, and liquid-in-glass thermometers, the uncertainty of these thermometers in calibration, and in use, is 

often poorly documented resulting in significant error in 

defining calibration interval requirements or sensor 

replacement. Both of these are very costly to many endusers. 

Recent performance evaluation tests of two types of industrial thermometers have been completed. Because 

thermocouples are often installed in elevated-temperature 

environments for long periods of service, we have recently focused on documenting the drift characteristics of 

these sensors at elevated temperatures. In one set of tests, 

the drift of common base-metal thermocouples was measured over one year at a temperature of 200 °C. The observed drift rates (as large as 0.3 °C over one year for a 

fixed installation depth, and as large as 1 °C for variable 

depth) are highly predictable. Thus, the results of this 

study can be used to establish periodic replacement intervals in industrial applications, eliminating the need for 

expensive, and often impractical, periodic calibrations. In 

another set of tests, we determined the drift of thermocouples during the act of calibration. This drift is a key 

component in the calibration uncertainty that is often neglected in industry, resulting in an unrealistically optimistic calibration uncertainty. For liquid-in-glass thermometers, previously published repeatabilities had no clear statistical basis. We recently completed a study of the repeatabilities and calibration uncertainties of a selection of 

18 liquid-in-glass thermometers over the range −20 °C to 

400 °C. The results demonstrated that the now-obsolete 

published repeatabilities were a factor of 2.5 too large. 

NIST methodology developed for testing of industrial 

thermometers is often adopted by industry, either by 

direct familiarity with NIST publications or by incorporation of NIST methods in consensus standards by 

ASTM or other organizations. The values obtained by 

NIST for both repeatability and drift are trusted in industry as an impartial assessment of expected sensor performance. Performance- testing results from well-conceived experiments provide a 

basis for rational change in their use and in the standards supporting 

such use. 

It is anticipated that the results of 

this work, and similar efforts in the 

future, will become the basis for 

changes in documentary standards 

issued by ASTM and other standards 

development organizations. 

The market for industrial thermometers is 

about $380B; therefore CSTL scientists are 

conducting an ongoing project in sensor characterization to support their effective use in 

both industry and government. 

Automated Carriage

Wells for 54

Thermocouples

Stirred oil bath for the determination of longterm drift of thermocouples at 200 °C. The carriage allows testing of thermocouples under 

continuous thermal cycling by automated insertion and withdrawal from the bath. 
101

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 NIST 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. For many decades, the NIST Thermometry 

Group has offered the Precision Thermometry Workshop at its Gaithersburg campus, and we continued this tradition in 2004. We have developed 

a number of additional training classes that complement the overview 

provided by the Precision Thermometry Workshop. At the 2004 Measurement Science Conference, we presented a newly developed workshop 

on the “Selection, Calibration, and Use of Contact Thermometers.” To 

provide more extensive hands-on training, we are developing a series of 

small workshops that provide intensive laboratory instruction on particular techniques, using the highest quality of 

equipment available at NIST. We have held the ITS-90 Fixed-Point Cell Mini-Workshop for several years, and last year 

a new workshop on Liquid-in-Glass Thermometers was added. 

Recently the number of laboratories seeking accreditation in the field of thermometry has significantly 

increased. Many of these laboratories claim uncertainties comparable to those of many National Metrology Institutes. Staff from the NIST Thermometry 

Group have increased our efforts in accreditation 

activities by a) serving as technical assessors, 

through the National Voluntary Laboratory Accreditation Program (NVLAP), of laboratories claiming 

low levels of uncertainty, b) creating guidelines of appropriate levels of proficiency testing, c) designing and conducting proficiency tests, and d) assisting companies in meeting accreditation requirements in foreign countries. The expertise of NIST staff is an invaluable resource for companies seeking accreditation, and our involvement helps to ensure that accreditation is a reliable indicator of quality. 

Because of our outreach activities, the members of the NIST Thermometry Group are a world-renowned source of 

thermometry expertise. Several secondary calibration laboratories in the US have attained a level of competence in 

thermometry that is commensurate with many foreign National Metrology Institutes, partly because of ready accessibility to methods established at NIST. Future outreach activities will address the specific needs of particular industrial sectors. For example, workshops previously held in conjunction with a conference on semiconductor processing and with 

the ASTM committee on petroleum have led to requests for preparation of training materials for use in these industries. 

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 highthroughput techniques, due to their extraordinary selectivity and sensitivity. These new analytical methods are becoming increasingly more 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 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 and intensity. These glasses are 

CSTL thermometry research is 

combined with outreach activities to 

provide increased visibility for our 

work, a mechanism for training and 

education of users, and a forum for 

cooperation and exchange of ideas 

with other scientists. 

The CSTL team will continue involvement with Standards Development Organizations such as IEC, 

ASTM, and ASME. 

CSTL researchers work to prepare, characterize, and certify a 

set of fluorescent glass SRMs 

that will enable the shape and 

intensity of spectral emission to 

be calibrated on fluorometers in 

the near UV and visible regions 

of the spectrum. 
102

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 photodegradation. 

Spectral emissivity standard candidates have 

been identified with UV/violet, blue, green 

and yellow/orange fluorescence, corresponding to CeO2, CuO, U3O8, and MnO2 dopants, 

respectively. At this point, the U3O8 and 

MnO2 glasses have been most thoroughly 

studied. They are both highly resistant to 

photodegradation and possess all of the desired characteristics for spectral emissivity 

standards. 

These standards are in the final stage of production and will be certified within the next 

year. They are being made in the form of a 

standard-sized cuvette with three polished 

long faces for 90-degree detection and one 

frosted long face for front-face detection. 

The CeO2 and CuO glasses have both been shown to have desirable 

spectral characteristics and are presently in the prototype stage of 

development. Once certified, the U3O8 and MnO2 glasses can be used in 

combination with SRM 936a quinine sulfate dehydrate to cover the visible region from 400 nm to 700 nm as shown in the figure. (SRM 936a is 

a blue spectral emissivity standard that is the only standard presently 

sold for spectral correction of fluorometers.) The CeO2 glass will be 

used to cover the near UV/violet from 350 nm to 450 nm. We hope to 

replace SRM 936a with the CuO glass, as the present standard is an organic dye that has many of the stability problems associated with organic fluorophores. Since all of these glasses are highly resistant to 

photodegradation, they can also be used as day-to-day intensity standards for instrument qualification. 

Molecular Simulation of Alkylsilane Stationary Phases in Liquid Chromatography 

K. Lippa and L.C. Sander (839)

Covalently modified surfaces represent a unique state of matter 

that is not well described by liquid- or solid-phase models. The 

chemical bond in tethered alkanes imparts order to the surface in 

the form of anisotropic properties that are evident in chromatographic and spectroscopic studies. An understanding of the 

structure, conformation, and organization of alkyl-modified surfaces is requisite to the design of improved materials and the 

optimal utilization of existing materials. Computational simulations offer insights into the structure of covalently modified surfaces that may not be apparent through empirical observation. 

0

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Wavelength (nm)

Relative Fluorescence Intensity

Ce

Cu

U 

Mn

SRM936a Spectral emission profiles of each 

of the four glasses in the suite of 

standards, compared with that of 

SRM 936a 

We are searching for a suitable MOD 

glass for a red emission standard that 

would cover the region from approximately 680 nm to 800 nm, and beyond. 

We are presently working with a 

nanocrystal manufacturer to help us to 

identify such a nanocrystal-based material. We are also working with the 

same manufacturer and a glass manufacturer to develop fluorescent thin 

films to be used as intensity standards 

for microarray readers. 

CSTL researchers develop models that 

clearly indicate the extent of molecular order within alkyl-modified surfaces and are 

consistent with spectroscopic studies. The 

results provide a compelling vision of alkylmodified surfaces that will guide future development of materials for chromatography and biotechnology. 
103

Simulations offer several advantages over physical experimentation. Models provide a means to visualize the important 

features of a surface at the molecular level in a way that is not possible by physical experimentation. Models can be 

precisely described in terms of bonding densities, chain placements, temperature, etc. The influence of changes in specific aspects of the model can be tested directly without altering other parameters. For example, alkyl chain length effects can be studied by extension of existing chains without changing their placement on the silica surface. Simulations 

can provide insights into the conformation and dynamic aspects of physical materials; however, the utility of the data 

depends on the suitability and accuracy of the computational approach employed. 

Molecular dynamics (MD) is a time-dependent simulation of molecular motion. Initially, velocities are assigned to atoms (often based on Boltzmann distribution) such that the net momentum is zero (i.e., no flow). After a finite time interval, the energy of the system is calculated. Forces on atoms are calculated, new velocities are determined, and the 

process is repeated. Coordinates are saved periodically, and analysis of the coordinates over time provides information 

on molecular motion. Monte Carlo approaches utilize random sampling to choose positions, orientations, etc. of molecules. The energy of each sample is calculated, and the new configuration is accepted or rejected. 

Molecular mechanics calculations are typically used to calculate the energy at intermediate steps. Molecular mechanics computations are based on algebraic relations that describe the energy of the molecule(s) in terms of stretching, bending, 

torsions, electrostatic and van der Waals forces, etc. These terms constitute the 

force field; any required constants are provided by experiment or ab initio calculation (parameterization). 

Investigation of Arsenic Oxidation-State Speciation in Primary Calibration Solutions and Effects on 

ICP-OES Measurements 

L.L. Yu, T.A. Butler, and G.C. Turk (839) 

It is well known that the toxicity of arsenic is strongly tied to oxidation 

state speciation. Less understood is the effect that arsenic oxidation state 

has on measurement accuracy. Standards prepared from arsenic metal, 

which in principle should yield a more accurate and precise standard 

relative to those prepared from an arsenic compound, were frequently 

found to have inexplicable disagreements between batches when analyzed by using optical emission or mass spectrometry. The deviation, 

which can at times be as great as a few percent, frustrates the efforts of 

producing an accurate clinical standard for arsenic. Our studies of the 

cause of this disagreement between the arsenic standards point to differences in arsenic valence states between solutions. The results of an investigation into the kinetics of the reaction for the arsenic standard preparation elucidated why arsenic solutions comprised of varied arsenic species were produced. Guidelines were developed based on the investigation for producing a 

single-species arsenic standard, thus eliminating the measurement bias in the certification process, ensuring the production of more accurate and precise arsenic standards for clinical and general use. 

We analyzed sets of standard solutions prepared by dissolving arsenic metal with nitric acid and found both arsenic III 

and arsenic V to be present in these standards, including various lots of SRM 3103a, the arsenic spectrometric solution. 

Furthermore, we discovered that standards containing greater amounts of As III yielded somewhat lower signals in ICP 

optical emission spectrometry than those in which the predominant form of arsenic was As V. Similar results have also 

been observed using ICP mass spectrometry. This difference in the instrumental response of these two forms of arsenic 

will result in a measurement bias if samples and calibration standards differ in the form of arsenic. The figure below 

shows the results of a study of the kinetics of the arsenic metal digestion process. In the experiment small aliquots of 

solution were periodically withdrawn over a period of 40 hours from a digestion vessel as a piece of arsenic metal was 

being dissolved in a solution containing a volume fraction of 50% of concentrated nitric acid in water on a warm hotplate. The withdrawn aliquots were analyzed using liquid chromatography with ICPMS detection to determine the fraction of the total dissolved arsenic present as As III. The results (see graph) show that at the beginning of the process 

most of the arsenic is present as As III, and all of the arsenic is present as As V after 25 hours. The discontinuities in 

the reaction curve occur when additional nitric acid / water is added. 

An extension of the simulation effort is planned to probe 

the effect that various solvent 

environments in contact with 

the alkyl-modified surface 

may have on alkyl chain conformation. 

CSTL researchers produce a highly 

precise and accurate arsenic standard by investigating and eliminating an unexplained bias between the 

gravimetric preparation value and 

the inductively coupled plasma optical emission spectrometric (ICPOES) value of arsenic standards. 
104

All of the metal appears to be dissolved after only 15 hours. If the process had been stopped after 15 hours, 

the resulting solution would contain a 

mixture of arsenic oxidation states. It is 

clear that this situation occasionally 

occurs in our production of arsenic 

solution standards, including SRM 

3103a. 

The result of the research resolves a long-standing mystery regarding bias sometimes observed between the gravimetric 

preparation value of certain arsenic solutions and values determined spectrometrically using independent calibration 

solutions. A revised digestion procedure was developed as a result of this research to ensure the production of a more 

accurate and precise arsenic standard for clinical and general applications. The research also raises some questions regarding the accuracy of arsenic measurements by using plasma-source-based spectrometry techniques. Further studies 

will determine if other elements are susceptible to similar systematic bias. The underlying cause of the bias in plasma 

source spectrometry remains unknown. 

New Series of Standard Reference Materials (SRMs) for Raman Intensity Correction 

S. J. Choquette (839), W. Hurst (836), and E. Etz (837) 

Raman spectroscopy is a nondestructive analytical method that has 

many qualitative and quantitative analytical applications in homeland 

security and forensics, as well as the pharmaceutical and biotechnology industries. Its major advantage over infrared (IR) spectroscopy as 

an analytical tool is that it may be used with little or no sample preparation. Because Raman scattering is a single beam emission process, 

the appearance of a compound’s Raman spectra is highly dependent 

on the wavelength of the excitation source and the construction of the 

spectrometer. As a result, Raman spectral libraries are only useful for the specific instrument configuration upon which 

the spectra were measured. The development of a simple-to-use emission standard would allow the user to correct the 

sample spectra for instrument response. Corrected spectra, much like absorbance spectra, would be transferable between 

instruments, would be amenable to searching in corrected libraries, and would allow verification of instrument performance. 

Currently three standards have been produced that are suitable for use with systems employing 785 nm lasers (SRM 

2241), 532 nm lasers (SRM 2242) and 488 nm/514.5nm lasers (SRM 2243). The development of the standards will 

allow a traceable calibration of the system response to NIST 

standards, which is a necessary step for qualification of 

chemical instrumentation in the pharmaceutical industry. 

We have developed a procedure 

using iodine as an oxidant to convert solution standards containing

mixed oxidation states of arsenic 

to As V, thus eliminating the 

source of bias. 

The CSTL research team developed 

a suite of inexpensive intensity correction standards for commercial 

Raman systems employing a number 

of laser excitation wavelengths. 

The use of these new SRMs will enable the creation of standardized Raman spectral libraries, an 

increasingly important task given the number of 

spectrometer vendors marketing this technique 

to first responders, forensic scientists, and military personnel. 

0%

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Digestion Time (h)

Kinetic Study of As metal digestion

Percent (%) of As III vs time (h)

All of the 

Metal 

digested 
105

This year we completed work on SRM 2243: a 

relative intensity correction standard for 488 

nm/514.5 nm systems, and initiated work on 

SRM 2244: a relative intensity correction 

standard for 1064 nm lasers. The latter is particularly important as our customer base for 

SRM 2244 is the pharmaceutical industry. FT 

Raman systems are widely used in this industry because of the large installed base of FTIR systems (similar instruments/software) and 

also the Raman spectra of compounds excited 

with NIR excitation tend to exhibit less fluorescence interference. We have identified a 

suitable candidate glass for SRM 2244 and 

have initiated a round robin study with a number of instrument vendors to assess compatibility and utility of the glass. Based upon favorable feedback concerning this glass, a production melt was procured and is currently 

being processed by our glass shops. We anticipate delivery of this SRM in FY 2005. 

AMDIS – Automatic Mass Spectral Deconvolution and Identification Software 

G. Mallard, S. Stein, and O. Toropov (838) 

One of the most powerful tools for the analysis of 

complex mixtures is the combination of the gas chromatograph (GC) to separate the mixture into component parts, followed by the mass spectrometer (MS) to 

identify each component as it is eluted. This combination GC/MS is the most widely used analytical tool for 

low-concentration analysis for food safety and environmental monitoring. In addition it is widely used in 

general organic analysis, the development of new flavoring agents, the analysis of fragrances, and in many 

medical applications. 

NIST’s Automatic Mass Spectral Deconvolution and 

Identification Software (AMDIS) was originally developed for detection of chemical weapons in complex 

mixtures such as might be found in the environment or 

in chemical process streams. It was designed to work 

without analyst input as a method of insuring that sensitive business information that could be present in a 

process stream was not compromised. 

-400000

-200000

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Cyclohexane Raman Spectra on Three Raman 

Spectometers Using 785 Excitation 

Raman Shift 

-.8

-.6

-.4

-.2

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3000 2500 2000 1500 1000 500 

Raman Shift

SRM 2241 Corrected Raman Spectra 

The top graph depicts the uncorrected 

Raman spectrum of cyclohexane acquired on three different Raman systems. All three spectrometers used 785 

nm laser excitation. As can be seen, the 

height of the bands is different with each 

spectrometer. The next graph shows 

those same spectra corrected with SRM 

2241. The band heights of the spectra 

are now similar. 

At extremely low concentrations, it can be difficult to extract the signal from the data file due to 

the very complex background that is present. 

The time spent by an analyst trying to analyze 

the data file can be many hours. AMDIS is a solution to this problem. 

Relative Intensity vs Raman Shift 

(wavelength, cm-1) 

Relative Intensity vs Raman Shift 

(wavelength, cm-1) 
106

The data that AMDIS analyzes, such as those 

shown above (which is a plot of total MS ion current as a function of elution time from a GC) are 

not typically the large obvious peaks, rather they 

are the very small components that are not obvious 

to the user. In this case, AMDIS extracted 341 distinct components from this data file (information 

provided by the components label in the figure). 

Typical software provided with the instruments 

might have found 50, and the analyst would have 

still had more work to do to identify the large majority of peaks. AMDIS can of course analyze the 

larger peaks, but the time spent by the analyst on 

these is small and in most cases the answer is 

known before the analysis is done. It is the small 

components that can change the flavor, can be the 

toxic residues, and can be the indicator of disease. 

It is in extracting these small components that 

AMDIS excels. Shown in the figure to the right (in 

the top window) is an enlargement of the region 

indicated by the red rectangle in the previous figure. This is the location of the caffeine found in the 

data file. In the second window is a mass spectrum 

from the region (in red box the elution time plot) where the caffeine was found. The signal in this region is very small, 

as indicated by the full-scale abundance in each window. Here the white lines (displayed next to the extracted peaks) 

indicate the portion of the data that was used for identification the caffeine. The bottom panel shows the extracted data 

(again in white) compared to the library spectrum of the caffeine. The entire process takes a few minutes rather than the 

hours that an analyst would take to do determination manually. 

In the last year the growth in the use of AMDIS by the 

organic analytical community has been very strong. One 

of the most exciting developments has been the incorporation of AMDIS into a new set of tools for automatic 

analysis developed by Agilent Technologies. The tools 

have been given the general name of Deconvolution Reporting Software (DRS) and incorporate Agilent Technologies run time locking technology, the NIST search 

software, and AMDIS in a combined tool. DRS allows 

users to identify pesticides more confidently and at lower 

concentrations and with more confidence than had been 

possible with the Agilent system alone. 

AnIML – Analytical Information Markup Language 

B.A. Schaefer (University of Kaiserlautern, Germany), D. Poetz (Fachhochschule Wiesbaden, Germany), 

A.D. Nguyen, 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 fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) systems can interchange their respective data. However, 

in the last few years, the emergence of platform- and applicationindependence through the Internet has advanced the possibilities for 

data interchange beyond the capabilities of these protocols. Today’s 

analytical chemists need not only to interchange data generated within a 

specific technique, they need to interchange, import, export, store, and 

In the above example (cardamom oil with very low levels of

pesticides) AMDIS found the pesticides, but also found low

levels of caffeine. 

Agilent Technologies incorporated AMDIS into 

a new set of tools for automatic analysis under 

the general name of Deconvolution Reporting 

Software. 

In collaboration with ASTM Subcommittee E13.15 on Analytical Data, 

we are creating an extensible markup 

language (XML) for analytical chemistry result data. Based in part on our 

previous SpectroML markup language for UV/visible result data, this 

ASTM effort is being called the Analytical Information Markup Language (AnIML). 
107

combine all their data from multiple sources at multiple sites. Users are now no longer content with just having data 

plots. They need real access to their data within a report – 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 who generate 

it. Proprietary data formats and existing interchange programs simply cannot fill such corporate needs for data accessibility 

Over the past three years, we developed an XML-based mechanism for instrument-to-instrument, instrument-to-application, and application-to-application 

data interchange called SpectroML: an extensible markup language for molecular spectroscopy data. SpectroML was created initially to serve as a model implementation of a markup language for molecular spectroscopy and ultimately 

to provide a web-based mechanism for interchanging UV/visible spectral data 

generated on different spectrophotometers with our optical filters database. 

When our optical filters database was created as a replacement for paper records, we could import data only from the High Accuracy Spectrometer (HAS) 

used to certify the SRM optical filters. The complex and archaic data importation process badly needed to be modernized, and we wanted to be able to import 

data from our other spectrometers as well. Using SpectroML drivers/translators for the HAS-II, the PerkinElmer 

Lambda 900, and the Hewlett-Packard HP 8453 spectrometers, data from these instruments can now be converted into a 

single format SpectroML file and imported into our optical filters database. SpectroML is an integral part of the process 

for certifying and recertifying optical filter SRMs; plus, we can import research data taken on machines other than the 

HAS for comparison purposes. 

Even before we developed SpectroML, we began to proselytize both instrument vendors and the 

ASTM E13 Molecular Spectrometry and Chromatography Committee about the merits of an 

XML-based approach to data interchange. In 2001, the ASTM E13 committee established a task 

group to develop a markup language for molecular spectrometry and chromatography, and in 

2003 the task group was made into a full ASTM subcommittee—E13.15 on Analytical Data. 

E13.15 has been working with instrument vendors and the IUPAC Committee responsible for 

JCAMP-DX to develop a unified XML-based approach for interchanging molecular spectrometry and chromatography data called the Analytical Information Markup Language (AnIML). 

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. The core 

schema defining the common notions of sample information, measurement data, system information, quality assurance, etc. will be handled by ASTM E13.15. Technique-specific base 

documents will build on the core and will be the responsibilities of the respective E13 subcommittees in conjunction 

with other interested expert groups such as those from the IUPAC, American Society for Mass Spectrometry, etc. 

Data/instrument vendors are responsible for implementations of the instrument/origin specific base document extensions that will build on both the core and the technique-specific standards. Higher-level specifications could include 

organization-specified extensions that are company-specific and even end-user or application-specific extensions. The 

core is being built as much as possible to bridge previous data exchange standards work (e.g., ANDI and JCAMP-DX), 

and efforts are being made to collaborate with and include other organizations with similar interests and as much as 

possible to reuse terminology and concepts from existing standards. 

Over the past year, the schemas for the core and technique definitions have been written and base documents for several 

techniques have been created. Since the metadata for individual techniques will be maintained in instance documents 

instead of schemas (to permit extension of technique metadata) the standard mechanism that applications use to validate 

XML documents syntactically cannot be used. Accordingly, we created a Technique Validator program that provides 

semantic and syntactical checking as well as some bounds/limits checking for AnIML files. We are now working with 

several groups of domain experts to develop AnIML. To solve the problem that most of the domain experts do not know 

XML, we have written a Technique Creator program that guides a user through the creation of a technique base document and then creates the XML code. 

Currently, work is underway to implement the AnIML core schema and to develop an example implementation of a 

technique-specific AnIML schema for liquid chromatography photodiode array spectra. If successful, we can begin implementing AnIML for other techniques. The IUPAC committee has already converted their JCAMP-DX terminology 

to XML so progress should be rapid for those techniques supported by JCAMP-DX. The AnIML project also dovetails 

nicely with another IUPAC project to convert the IUPAC units and terminology documents (Green and Gold books) to 

XML. 

AnIML 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. 
108

XML for Microanalysis 

J. H. J. Scott (837) 

The rapid spread of XML technology in many fields of chemical analysis has led to renewed interest in applying XML to the data exchange 

and data storage problems faced by the microanalysis community. Information about all aspects of a microanalysis experiment (not just 

spectral results) can be captured by a well-designed Microanalysis 

Markup Language (MML), which should: 

• Enable the free exchange of spectral information among microanalytical instrument users by developing an XML-compliant markup language for spectroscopy data. 

• Provide a framework for sharing microanalysis data community-wide to allow more sophisticated 

theoretical model building and model assessment to take place. 

• Create a document format for long-term archiving of microanalysis data that diminishes the effect of “bit-rot” and software decay and helps prevent the loss of data due to lost file formats. 

After testing several variation of the markup language and exploring design tradeoffs, as well as implementing multiple 

working prototypes to store test data, we began to integrate the microanalysis XML effort into the ASTM E13.15 

AnIML, which is the analytical data markup language project also CSTL-led but by an independent team as described in 

the previous article. The two projects can interoperate cleanly and a microanalysis component can be added to the 

AnIML project through the development of technique schemas (defined by the AnIML framework) based on common 

microanalysis data acquisition modalities such as scanning transmission electron microscope (STEM), scanning electron 

microscopy (SEM), and elemental analysis spectroscopies such as electron energy-loss spectroscopy (EELS) and energy-dispersive x-ray spectrometry (XEDS). 

This year we implemented multiple working prototypes of 

Microanalysis Markup Language (MML) based on the original ASCII file format created in 1991 by the Electron Microscopy Society of America (EMSA) task force, currently the 

Microscopy Society of America (MSA). The larger CSTL 

team began to integrate concepts from SpectroML and 

AnIML, and began contributing to AnIML development by 

attending meetings, studying the schema and framework, and 

learning the role of ASTM Analytical Data Sub Committee 

E13.15. We estimate that the creation of new microanalysis 

technique schemas for AnIML covering spectroscopies and 

electron optical scanning techniques (STEM, SEM, Spectrum 

Imaging) will add significantly to their schema list while the 

XML-based metadata is already useful for storing information 

about spectrum imaging experiments to complement LISPIX 

.rpl files and 3D Chemical Imaging competence. 

In the coming year we plan to extend and refine the standard to resolve remaining questions while developing a more 

detailed model for XML use in spectrum imaging applications. We would also like to explore the use of XML in building a data pipeline for 3D Chemical Imaging at the Nanoscale. 

CSTL researcher creates new microanalysis technique schemas for 

AnIML covering spectroscopies and 

electron optical scanning techniques 

(STEM, SEM, Spectrum Imaging). 

#FORMAT : EMSA/MAS Spectral Data 

File 

#VERSION : 1.0 

#TITLE : NIO EELS OK SHELL 

#DATE : 01-OCT-1991 

#TIME : 12:00 

#OWNER : EMSA/MAS TASK FORCE 

#NPOINTS : 20. 

#NCOLUMNS : 1. 

#XUNITS : Energy Loss (eV) 

#YUNITS : Intensity 

#DATATYPE : XY 

#XPERCHAN : 3.1 

#OFFSET : 520.13 

#CHOFFSET : -168 

#SIGNALTYPE : ELS 

#XLABEL : Energy 

#YLABEL : Counts 

#BEAMKV -kV: 120.0 

#EMISSION -uA: 5.5

<?xml version="1.0"?> 

<spectrum xunits="Energy Loss (eV)" 

yunits="Intensity" signaltype="ELS"> 

<title>NIO EELS OK SHELL</title> 

<date>1991-10-01</date> 

<time>12:00</time> 

<owner>EMSA/MAS TASK FORCE</owner> 

<xperchan>3.1</xperchan> 

<offset>520.13</offset> 

<choffset>-168</choffset> 

<beamkv>120.0</beamkv> 

<emission-uA>5.5</emission-uA> 

<probecurrent-nA>12.345</probecurrentnA>

Example file excerpts from the headers of the existing 

EMSA/MAS Spectrum File Format 1.0 (top), and Microscopy Markup Language (MML), a proposed XMLbased enhanced format (bottom). Both formats are designed to be human-readable/editable and intuitive instead of efficient and parsimonious. The XML-based 

format has the added advantages of being extensible, 

web-aware, more easily validated, and more tolerant of 

vendor customization. It is also backed by a large, professionally-developed codebase, a suite of tools, and 

plentiful documentation on XML itself. 
109

Properties and Processes for Cryogenic Refrigeration 

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

Cryogenic temperatures are required for many technology areas, including infrared sensors for surveillance and atmospheric studies; superconducting electronics, 

magnets, and power systems; to create clean vacuums in semiconductor fabrication processes; for liquefaction of industrial gas; and many other existing and future applications. Proper measurements need to be established to characterize 

losses within cryocoolers and models need to be developed to optimize the design 

of such systems. Material properties at cryogenic temperatures also are needed by 

industry for the design of cryogenic equipment, but the data are difficult to find 

and interpret. 

This year, we redesigned our web site to provide 

clear access to references for the database on 

cryogenic material properties and to graph each property as a function of temperature between about 4 K and 300 K. In 

addition, we developed two improved measurements techniques (based on hot-wire anemometers) that give good agreement for oscillating flow and used them to measure the complex flow-impedance of an inertance tube. As part of a 

CRADA with a medical device company, we completed measurements on the performance of a simple pulse tube refrigerator for dermatological pens to about –90 °C (such operating temperatures are higher than those normally achieved 

with pulse tube refrigerators). Using a compressor with a piston-position sensor we were able to characterize the overall 

system performance for this device. 

In the future, we will continue expanding and improving the 

cryogenic materials database, and developing test methods and 

improvements in our cryocooler simulation models to address 

fundamental limitations to the efficiency of cryocooler systems. 

A comprehensive article on the fundamentals 

of cryogenic refrigeration for both small and 

large superconducting systems was published:

“Refrigeration for Superconductors,” IEEE 

Proceedings on Electronic and Large Scale 

Applications of Superconductivity. 

NIST scientists address 

issues associated with cryogenic refrigerators, i.e., 

cryocoolers, and materials 

performance that are limiting growth in all these 

technology areas. 

http://cryogenics.nist.gov/NewFiles/material_properties.html
110
111

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 US microelectronics manufacturing globally competitive. 

Nanofabrication of Test Architectures for Molecular Electronics Applications 

J. D Batteas, J.C. Garno (837), C.A. Zangmeister (836) 

The ability to precisely construct nanoscopic metal-molecule-metal junctions is of great importance for the development of robust molecule-based 

electronic and optoelectronic devices. By combining scanned probe lithography with electroless metal deposition, metal-molecule-metal junctions can 

be constructed where the dimensions and surface organization are dictated 

by the placement of surface groups active for electroless deposition, such as 

carboxylates, into a desired pattern. These structures will provide a means of 

reproducibly measuring transport (current-voltage) properties of ensembles 

of molecules with a fixed a real density to assess the role of the molecule in 

the transport process and to determine the scalability of per molecule conduction in such junctions.

The CSTL research team developed methodologies to construct confined molecular assemblies of metal-molecule-metal 

junctions with nanoscale dimensions for the evaluation of electron transport in small ensembles of molecules (10’s to 

1000’s) to determine the scalability of per molecule conduction values in prototypical device architectures. To this end, 

automated scanned probe lithography is employed to pattern surfaces with self-assembled monolayers, where specific 

molecular linking groups are placed into organized structures on Au surfaces with features sizes ranging from approximately 10 nm to 200 nm in lateral dimension with the Au surface functioning as a bottom electrode. A completed metalmolecule-metal junction is then formed by the electroless deposition of metal yielding the top electrode. Here we have 

developed and demonstrated the needed fabrication capabilities to enable construction of these test architectures. 

Patterning of metal-molecule-metal junctions with nanoscale dimensions, specifically Cu on carboxylic acid terminated 

SAMs on Au, has been demonstrated using a combination of automated scanned probe nanofabrication and electroless 

metal deposition. The architecture of the nanostructures formed can be precisely tuned in all dimensions by controlling 

the lateral 2-D pattern size of the fabricated structures, the surface density of reactive acid groups placed into the structure, and the concentration of reactants in the electroless metal plating solution. An overview of the key steps to fabricate the Cu nano-contacts is illustrated in the figure below. Using this approach metal structures ranging from approximately 20 nm to 200 nm in lateral dimension with 5 nm to 10 nm metal top contacts have been formed. 

The value of the US Semiconductor Manufacturing Equipment 

industry is $15B, and the world 

market is about $50B. Wafer 

fabrication equipment accounts 

for about 70% of the market. 

The CSTL research team develops a robust, systematic nanoscale test structure to critically 

evaluate electrical properties of 

candidate materials that are used 

in the development of nanoscale 

molecular-based electronic devices. 

A B

C

Au(111) Au(111)

Au(111)

- OH

-S

-COOH

Cu

AFM

tip

Cu plating

solution

A B

C

Au(111) Au(111)

Au(111)

- OH

-S

-COOH

Cu

AFM

tip

Cu plating

solution

Scheme for fabrication of nanoscale 

metal junctions. 

A. First a resist layer is deposited on the surface. 

B. A structure of 16- Mercaptohexadecanoicacid 

(MHA) is then patterned into the resist by nanografting. 

C. The surface is then exposed to a Cu plating 

solution yielding C. 

* AFM = Atomic Force Microscopy 
112

An array of final structures fabricated using this approach is shown in the figure to 

the right. The patterning process requires only on the order of 30 min for forming 

all of the junctions. This provides a means a fabricating a range of structures with 

varying dimensions to assess scalability of current with junction dimension and 

molecular packing density. This approach should be readily extended to other metals that can be electrolessly deposited such as Pt, and be applicable to the construction of more complicated multidimensional metallic structures including combined 

vertical and lateral junction geometries. Additionally, well-defined nanoscale 

metal-metal junctions can be also formed which may be employed in the development of enhanced optical spectroscopic methods for sensing. 

Current experiments are now aimed at probing the electrical behavior of ensembles 

of molecules within the nano-patterned junctions. These measurements can then be 

compared to those from prototypical device structures. This approach for building 

nano-patterned metallic structures will also be transferred to Si substrates as a 

means to construct hybrid semiconductor-molecule-metal junctions. 

Horizontal Growth and In Situ Assembly of Oriented Zinc Oxide Nanowires 

B. Nikoobakht and S.J. Stranick (837) 

Details provided in the Measurement Science for Future Standards and Technologies Section of this report. 

Thermophysical Properties of Gases used in Semiconductor Processing 

J.J. Hurly, K.A. Gillis, and M.R. Moldover (836) 

The semiconductor industry identified the highest-priority 

process gases, “surrogate” gases, and the binary mixtures 

of process and carrier gases as well as targets for accuracy 

required to model manufacturing processes. 

CSTL provides the data for the speed of sound, ideal gas 

heat capacity, viscosity, thermal conductivity, and the virial 

coefficients B(T) and C(T) that determine the pressuredensity-temperature relation for more than 20 of these 

process and surrogate gases. We obtain the thermophysical 

properties by exploiting novel, accurate, NIST-developed 

acoustic resonators. 

After installing a Greenspan viscometer in a facility for 

handling hazardous gases, this year we used it to measure 

the viscosity and the speed of sound of 12 process and 

“surrogate” gases under the conditions listed in the enclosed 

table. As in our previous work, the speed of sound was used 

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%. 

Topographic AFM image of 

nano-patterned array of sixteen 150 nm x 150 nm Cu 

structures (about 5 nm high) 

on carboxylic acid terminated 

SAMs. 

The components of a generic mass flow controller (MFC) and the thermophysical properties required to model them. 

Acquired data are disseminated to our customers via 

our web site http://properties.nist.gov/semiprop. 

The semiconductor process industry uses CSTLgenerated gas property values to model chemical 

vapor deposition (CVD) and to calibrate mass 

flow controllers (MFCs) that deliver process gases. 
113

The results of this research have been disseminated through publications 

in professional journals and by a series of talks at professional meetings. 

In the coming year, we will deploy a new monel spherical resonator and 

use it to measure the speed of sound in HF and SiF4 to determine the “best in the world” equation of state for these 

difficult-to-handle gases. We also expect to measure the viscosity of four hazardous process gases: SiF4, C4F8, CO, and 

CO2. We anticipate that these results will decrease the viscosity uncertainty for these gases from about 10% of value to 

approximately 0.5%. 

In Situ Calibration of Lightpipe Radiometers for Rapid Thermal Processing Between 300 °C to 700 °C 

K.G. Kreider, W.A. Kimes, and D.C. Ripple (836); B.K. Tsai (PL) 

Accurate temperature measurements are critical in rapid thermal 

processing (RTP) of silicon wafers for thermal oxidation and 

dopant anneals. Many RTP tools use lightpipe radiation thermometers (LPRTs) to measure the wafer temperatures during processing. 

These LPRTs can yield accurate temperature measurements when 

they are calibrated in situ or calibrated ex situ and used with the aid 

of a suitable model to correct for surface emissivity and chamber irradiation effects. Recent developments of LPRT sensor technology have enabled measurements to temperatures below 300 °C, compared to their previous lower limit of 

500 °C. Use at these lower temperatures requires extreme care due to the relative increase in background irradiation 

compared to the weak signal produced by Planck 

emission from the wafer itself. This work seeks to 

understand the metrology of in situ calibrations of 

LPRTs in this temperature region. 

NIST has assessed the performance of commercial 

LPRTs and their calibration and temperature measurement uncertainty in RTP tools. We developed 

highly accurate blackbody calibration techniques for 

the LPRTs; characterized and measured the temperaGases 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 

BCl3 300 - 400 1.3 

Cl2 280 - 400 3.2 

HBr 225 - 400 3.3 

-30

-20

-10

0

10

300 400 500 600 700

Temperature / °C

(Thin Film – CLRT) / °C

Wafer 1: No Spacer

Wafer 2: No Spacer

The graph indicates the difference between 

wafer temperatures as measured by the NIST 

thin-film thermocouple wafer and as measured 

with a cable-less lightpipe radiometer (CLRT).

The NIST team seeks to understand 

the metrology of in situ calibrations of 

lightpipe radiation thermometers in 

the range from 300 °C to 500 °C. 

J.J. Hurly,“Thermodynamic Properties of Gaseous Nitrous Oxide 

and Nitric Oxide from Speed-of-Sound Measurements,” Intl. J. of 

Thermophysics 24, (2003). 

J.J.Hurly, K.A. Gillis, J.B. Mehl, M.R. Moldover, “The Viscosity of Seven Gases Measured with a Greenspan Viscometer,” Intl. 

J. of Thermophysics 24, (2003). 

J.J. Hurly, Technical Editor of the Gases and Facilities 

Standards Committee of Semiconductor Equipment and 

Materials International, was honored for his “outstanding 

contributions” to the committee. 

The research team used a 

Greenspan viscometer to measure 

the speed of sound and viscosity 

of gases of importance to the 

Semiconductor Industry over a 

wide range conditions. 
114

ture sensitivity of the LPRTs; developed technology for in situ calibration of the LPRTs in the RTP tools using the 

NIST patented thin-film thermocouple calibration wafer; and measured the effects of wafer emissivity and LPRT proximity on the LPRT measurements in the RTP tool. 

New commercial LPRTs couple the optical detector directly to the lightpipe resulting in improved detectors for low 

levels of radiance. These CLRTs are capable of measuring temperatures below 300 °C. Typical results of calibrating a 

CLRT against the NIST thin-film thermocouple (TFTC) calibration wafer from 315 °C to 700 °C in the NIST RTP test 

bed are shown in the figure. Below 550 °C, light leakage from the heating lamps 

of the RTP tool introduced a significant error in the LPRT readings. By measuring the transient response of the LPRTs following rapid energizing of the heating lamps, we were able to differentiate between the radiance of the wafer and 

ambient chamber light. This allowed us to correct for the ambient chamber light 

from the radiance of the wafer. Our results, which reported the uncertainties 

attainable for in situ calibrations with and without correction of chamber irradiation effects, were published and shared with the semiconductor fabrication industry. 

The work that will follow will focus on the development and characterization of sensor technology for in situ calibration 

wafers. We are developing resistance sensors for use in the range 300 °C to 650 °C, where RTP tools are used in the 

formation of silicides and existing reference sensors are either fragile or unstable. We are also evaluating the sensitivity 

of commercial calibration wafers to perturbing heat fluxes. 

Chemical Characterization of SiGe Single-Crystal Specimens and SiGe Films on Silicon with Electron 

Probe Microanalysis 

R.B. Marinenko, D. Klinedinst, L. Richter, D. Simons, S. Turner, J.A. Small, E. Steel (837), and 

F. Stevie (NC State) 

The research team investigated three aspects of SiGe thin-films in 

an effort to prepare reference materials of SiGe on Si. First we 

studied thin films of SiGe on 8-inch diameter Si wafers for heterogeneity using wavelength dispersive electron probe microanalysis 

(WD-EPMA). Twelve 2 cm x 2 cm specimens obtained from 2 

different wafers were studied. The nominal compositions of the 

two films were 0.0384 and 0.341 mass fraction Ge, respectively; 

their approximate thickness was 95 nm and 170 nm, respectively. Secondary-ion mass spectrometry (SIMS) and elipsometry revealed a circular halo-like region around the disk center that was thicker than for the rest of the disk thus suggesting specimen-to-specimen heterogeneity. With this knowledge, specimens were selected from a region in the wafer 

where the specimen-to-specimen thickness variation was expected to be minimal. Using a point beam – taking readings 

on 10 points per specimen – the extent of the point-to-point heterogeneity within each specimen and between specimens 

was assessed. The expanded uncertainty, including all heterogeneity contributions, was estimated to be no larger than 

2.5% for Ge in 10 specimens. 

A heterogeneity assessment was conducted using WD-EPMA of five specimens cut from each of the SiGe14 and 

SiGe6.5 standard boules which were previously assessed for heterogeneity in 2003. The Ge in these five specimens had 

been analyzed with instrumental neutron activation analysis (INAA) and a sixth specimen had been analyzed by inductively-coupled plasma optical emission spectrometry (ICP-OES). We determined that the same sample-to-sample trends 

in the Ge composition shown by INAA were observed by WD-EPMA. Data, including backgrounds, were acquired at 

three different excitation potentials to enable quantification and evaluation of matrix correction procedures. This evaluation is ongoing and seeks to compare its results with those obtained using INAA and ICP-OES. 

SiGe films on Si (approximately 4 μm thick) were prepared by ASM America. 

Given their thicker profile it is be possible to treat them as bulk specimens with regard to quantitative electron microprobe analysis. Four specimens cut from two different disks with nominal compositions of SiGe10 and SiGe25 were tested using 

traverses and duplicate readings on random points to assess the heterogeneity. In 

addition, we acquired data at three excitation potentials with background readings for 

quantification. Using all these data we found that the expanded uncertainty (k=3 or 99% confidence interval) is less than 

1% relative for both Si and Ge, which is only slightly greater than the counting statistics error predicted from Poisson 

statistics. Currently, evaluation of the random point heterogeneity data and the quantification procedures are in 

The information provided by 

the NIST team will assist 

manufacturers and users of 

RTP tools to significantly improve the uncertainty of their 

temperature measurements. 

The CSTL-led research team provides 

the semiconductor industry with a reference material for evaluation of SiGe 

components. 
115

process. Data from the standard boules will be used in conjunction with 

Monte Carlo calculations to determine the optimum voltage and corrections procedures for microprobe analysis of the thicker films. We will 

also continue to evaluate procedures for the quantitative electron probe 

analysis of the thinner films discussed in the first part of these studies.

*Interactive Materials are materials that are donated by interested parties and distributed by NIST to private sector 

analysts. Data is submitted to the NIST IM Website, and evaluated by NIST staff that post collective results. This approach was designed to quickly make new materials (with some pedigree and evaluation) available to a specific community. 

Non-Contact Free Carrier Density Measurements for Compound Semiconductors 

J.E. Maslar and W.S. Hurst (836) 

The transport of free carriers is central to the operation of all 

optoelectronic devices and thus, reliable measurements of the 

carrier properties are critical. Hall or capacitance-voltage 

measurements have been traditionally used to obtain this information, but they require electrical contact, which precludes 

their use in situ during growth, processing, and 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. 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 Raman spectroscopy, including the semiconductor under investigation, the measurement system parameters, and the Raman spectral model used to fit the measured 

spectra. 

Materials with a range of properties suitable for use in a variety of optoelectronic devices operating at different regions 

of the electromagnetic spectrum are being investigated. Our primary focus is on narrow band gap group III-antimonide 

materials and wide band gap group III-nitride materials. Spectroscopic systems were optimized for each material system 

after examining samples provided by various collaborators (thin films of narrow band gap GaSb, GaAsSb, GaInSb, and 

GaInAsSb; wide band gap GaN, and AlGaN). Modeling of the Raman spectra from the different materials requires different spectral models that account for the differences in physical properties that make these materials suitable for different applications. Spectral models have been developed for n-type doped GaSb, p-type GaSb, n-type GaInAsSb, and 

n-type GaN. Efforts are underway to compare the results of the spectral models with electrical measurements to determine the suitability of the respective model. 

CSTL plans to release the materials 

for distribution as “Interactive Materials”* during the first half of FY05. 

The Raman spectra of ntype doped GaInAsSb epilayers shows a strong dependence on carrier density. 

The carrier density determined from the Hall effect 

measurement is indicated. 

The spectra are offset on the 

vertical scale for clarity. 

The results obtained by CSTL researchers 

should facilitate the use of Raman spectroscopy for spatially-resolved, off-line characterization as well as for process monitoring 

and control during film growth and subsequent patterning processes. 

200 300 400 500 600 700

nHall ≈ 3.3 x 1018 cm-3

nHall ≈ 2.0 x 1018 cm-3

nHall ≈ 4.6 x 1017 cm-3

nHall≈ 2.8 x 1017 cm-3

nHall ≈ 9.5 x 1017 cm-3

Unintentionally doped (/2)

Intensity (arb. units) vs 

Raman Shift (cm-1) 
116

Atomic Layer Deposition (ALD) – Process Models and Metrologies 

J.E. Maslar, E. Moore, and W.S. Hurst (836); D.R. Burgess, Jr. (838) 

Atomic layer deposition (ALD) is increasingly being used to deposit the thin 

(nanoscale) conformal layers required by many microelectronics applications, 

including high κ gate dielectric layers, diffusion barrier layers, copper seed 

layers, and DRAM dielectric layers. However, significant developmental issues remain for many of these applications. 

A solution to some ALD developmental issues might reside in technology 

computer-aided design (TCAD), which was identified by the 2003 International Technology Roadmap for Semiconductors as “one of the few enabling 

methodologies that can reduce development cycle times and costs”. Yet many 

difficult challenges to development of validated, ALD process models that 

allow prediction of equipment influences on film properties have been identified, including chemical data (e.g., rate constants, cross sections, surface 

chemistry), reaction mechanisms, and reduced models of complex chemical 

reactions. In addition, due to a lack of fundamental physical and chemical 

data of sufficient accuracy, experimental validation is a “key difficult challenge across all modeling areas” and the “major effort required for better model validation is without doubt sensor development”. This project seeks to assist in addressing some ALD developmental issues by providing validated, predictive process models and associated metrologies.

This work involves two mutually-supporting 

directions: (1) the development of in situ metrologies sensitive to ALD chemistry and (2) 

development of ALD chemical reaction 

mechanisms. We expect that experimental 

results that elucidate ALD chemistry will aid 

in chemical mechanism development and ultimately in process model validation. Further, 

the most important reaction species will be 

identified as understanding of a particular 

ALD reaction improves, thus facilitating the 

design of improved process metrologies. Ultimately, aspects of both the metrology and 

reaction mechanism development will be required to create validated ALD process models. ALD process models will be created by incorporating NIST’s chemical reaction mechanisms into commercial computational fluid dynamics codes and validating the resulting model under a range of parameters using experimental data 

collected during the course of this project. 

Optically accessible ALD reactors have been constructed for in situ, high-sensitivity Raman and infrared absorption 

spectroscopic measurements. Hafnium oxide films will be deposited from tetrakis (dimethylamino) hafnium and water. 

A two-dimensional numerical model was developed to simulate the flow of gas and temperature of surfaces in the 

experimental ALD reactors. Chemical models of HfO2 and Al2O3 ALD process chemistry are being developed and will 

be incorporated into the two-dimensional numerical reactor model. 

During the upcoming year, ALD recipes will be optimized for our reactors. Then, during actual HfO2 ALD, optical 

measurements will be performed on the deposition surface to determine the sensitivity of the optical measurements to 

various species present during ALD. Concurrently, we will search for correlations between specific growth steps uncovered by the more sophisticated optical methods with signals from conventional process monitors, such as a quartz crystal microbalance and mass spectrometer. 

Simulation of the flow in the ALD reactor. Chamber pressure =

133 Pa (1 Torr); inlet gas flow rate = 200 standard cubic centimeters per minute; wafer surface temperature = 572 K. 

CSTL researchers seek to 

develop in situ metrologies 

sensitive to Atomic Layer 

Deposition chemistry, and 

chemical reaction mechanisms leading to process 

model validation. 
117

In-situ Monitoring of Ion Bombardment Energies in Plasma Etchers for Improved Wafer Yield and 

Throughput 

M. Sobolewski (836) 

During plasma etching silicon wafers are bombarded by reactive chemical 

species and energetic ions, resulting in selective removal of material from 

exposed areas of the wafer. The energy of ions striking the wafer surface 

plays an important role in determining the etching rate, the profile of etched 

features, and the extent of plasma-induced damage. To obtain the best results, the kinetic energy of ions must be carefully optimized and controlled. 

Unfortunately, control of ion energy in industrial reactors is difficult because reliable methods for in situ monitoring are 

lacking. Traditional methods for measuring ion energy, which require that an ion energy analyzer be inserted into the 

plasma, are not compatible with industrial reactors. Typically, an analyzer will not survive very long when exposed to 

industrial plasmas, and it may cause contamination of the wafers being processed. Also, analyzers do not measure the 

ion energy at the most relevant location, the wafer surface. 

A CSTL researcher has developed and demonstrated a method for noninvasive monitoring of ion kinetic energy. It does not require that anything be 

inserted into the reactor. Instead it relies on measurements of the applied radio-frequency current and voltage, which are easily measured outside the reactor without any perturbation to the plasma or the process. These measurements are analyzed using electrical models of the plasma to determine the ion 

flux and ion energy distribution at the wafer surface. 

The method has been validated by comparisons with invasive ion energy 

measurements, and has recently been used to monitor drift in an inductively 

coupled plasma reactor. Over time, an electrically conductive layer builds up 

on insulating surfaces inside the reactor. This layer interferes with the operation of the inductive plasma source, causing a long-term downward drift in 

plasma density and ion flux. These changes in turn produce drifts in ion energy, which were monitored using the noninvasive technique. Depending on 

conditions, ion energies can either increase or decrease. In one experiment a 

change larger than 100 electron volts was detected. Such large changes in ion 

energy would certainly have deleterious effects on wafers being processed. 

The technique developed in CSTL has provided a better understanding of drift mechanisms that could further help process technologists and equipment engineers to minimize and eventually eliminate ion energy drift. 

CSTL researcher has developed a 

noninvasive method for monitoring the ion kinetic energy during 

plasma etching. 

Ar+ ion energy distributions obtained from 

noninvasive electrical measurements before (blue) 

and after (red) deposition of a conductive film on 

the inductive source window, for 1,000 Pa 

(10 mTorr) argon plasmas at 100 W inductive 

source power. (a) constant bias power at 1.0 MHz, 

and (b) constant bias voltage at 10 MHz. 

(a)

before

after

0 100 200

ion kinetic energy (eV)

ion flux

before

after (b)

0 50 100

ion kinetic energy (eV)

ion flux

(a)

before

after

0 100 200

ion kinetic energy (eV)

ion flux

before

after (b)

0 50 100

ion kinetic energy (eV)

ion flux

The non-invasive monitoring 

technique now enables early 

detection of ion energy drift so 

that response strategies can be 

implemented to prevent costly 

wafer damage. 
118

In-Situ Characterization of Additives Governing Copper Electrodeposition 

M.L. Walker, L J. Richter (837), and T.P. Moffat (MSEL) 

One of the critical issues facing the microelectronic/semiconductor industry is the robust 

fabrication of “on-chip” interconnections (wiring) between transistors and related devices. 

Due to its lower resistivity and superior electromigration characteristics, copper has replaced aluminum as the metal of choice for such interconnects. Interconnections are formed by electrodepositing copper 

into lithographically-defined sub-micrometer wide features (trenches and vias). The feature filling process occurs by 

superconformal or ”bottom-up“ growth that derives from a competition between electrolyte additives, such as inhibitors 

and catalysts, for available surface sites. A representative additive package to plating baths contains polyethylene glycol (PEG), chloride (Cl-

) and sulfonate-terminated disulfides such as Na2[SO3(CH2)3S]2 (SPS). Inhibition is provided by 

PEG/Cl-

 while disruption of the PEG species by SPS/Clleads to acceleration of the metal deposition. Incorrectly 

formulated baths can lead to uncontrolled electrodeposition, resulting in faulty interconnect creation and poor 

device performance. The PEG/Cl-

 system was studies to 

more fully characterize synergies between bath components and probe the utility of spectroscopic ellipsometry 

(SE) for the in situ evaluation of electrodeposition. 

The formation of a film upon the addition of PEG and Clwas observed, and the results indicate that in-situ dynamic SE can accurately measure film thickness with 

better than 0.1 nm accuracy under certain conditions. 

The results show the presence of Cl-

 is necessary for the 

formation of a film, but a film eventually forms regardless of additive order. The adsorbed PEG film thickness 

is approximately 0.5 nm, consistent with a single layer of 

helically-wound PEG. Additional studies on gold and 

silver substrates established that chelation of copper ions 

is not necessary for film formation. 

Studies on the three-component PEG/Cl-

/SPS system are 

planned to build on the insights gained from the PEG/Clsystem. Additionally, the use of other materials such as 

ruthenium as substrates for copper electrodeposition applications will be examined. 

NIST researchers adapt a commercially available spectroscopic ellipsometer to evaluate the effect of additives on 

metal substrates in liquid-based environments to mimic the 

electrolytic plating baths used in the industry. 

Ellipsometrically-determined model thickness 

trace of the layer formed on a copper substrate in 

the presence of the additives of PEG and Cl-

 in an 

electrolytic bath at a plating potential. In the top 

graph baseline was established at “1”, Cl-

 added 

after 4 minutes and measured at “2”, PEG was 

added at 10 minutes and measured at “3”. 

In the second graph the baseline was established at 

1, PEG added after 1 minute and measured at “2”, 

Cl-

 added after 2 minutes and measured at “3”. 

Spectroscopic ellipsometry is a nondestructive optical technique capable of 

measuring optical characteristics of a 

surface, and is routinely used in the 

semiconductor industry to evaluate wafer quality. 

1.0

0.5

0.0

Thickness (nm)

0 5 10 15

Time (min)

1

2 3

Cl- added first, 

followed by PEG

1.0

0.5

0.0

Thickness (nm)

0 5 10 15

Time (min)

1

2 3

1.0

0.5

0.0

Thickness (nm)

0 5 10 15

Time (min)

1

2 3

Cl- added first, 

followed by PEG

1.0

0.5

0.0

Thickness (nm)

0 5 10

Time (min)

1 2

3

PEG added first, 

followed by Cl1.0

0.5

0.0

Thickness (nm)

0 5 10

Time (min)

1 2

3

1.0

0.5

0.0

Thickness (nm)

0 5 10

Time (min)

1 2

3

PEG added first, 

followed by ClM.L. Walker, L.J. Richter, and T.P. Moffat, “In-Situ 

Ellipsometric Study of PEG/Cl- Co-adsorption on Cu, 

Ag and Au”, submitted to J. Electrochemical Soc. 
119

Characterization of Silicon Semiconductor Electronics Using SIMS Backside Depth Profile Analysis 

E. Windsor, G. Gillen, P. Chi, D. Bright (837), and J. Bennett (International SEMATECH) 

As silicon semiconductors continue to shrink, greater demands are placed 

upon the metrology techniques used to characterize these materials. If 

downscaling proceeds according to the schedule outlined by the International Technology Roadmap for Semiconductors, many metrology methods in current use will not have sufficient resolution to characterize future 

generation semiconductors. We envision that backside sample preparation 

combined with novel instrumental techniques such as C60+

 cluster ion 

bombardment will extend the utility of secondary-ion mass spectrometry 

(SIMS) measurements to the characterization of future-generation silicon 

semiconductors. 

This research team is working together 

with the Office of Microelectronic Programs (OMP) at NIST to develop metrology techniques for the characterization of 

future-generation semiconductors. SIMS is a useful characterization 

tool because the technique has high analytical sensitivity and good depth resolution. State-of-the-art SIMS instruments 

are capable of reaching sub-micrometer depth resolution. Such resolution, however, is often not achieved when analyzing semiconductors due to limitations imposed by the samples themselves. This is particularly evident in the characterization of shallow dopant profiles and the investigation of diffusion of material through thin gate dielectric films. For 

both these examples, the analysis area is the ultra-shallow region of the silicon substrate. This region is buried below 

numerous layers of different materials that were deposited during the 

manufacturing process. Using SIMS to analyze the area of interest, one 

must first sputter through these different processing layers. Depth resolution is degraded while sputtering through these layers because of the development of surface topography 

(especially when sputtering through 

polycrystalline materials) and the 

occurrence of “knock-on” ion beam 

mixing. With backside depth profile 

analysis, high depth resolution is 

maintained by analyzing the sample 

from the back (substrate) side rather 

than the front side. This eliminates 

the need to sputter through the multiple processing layers on the front side 

of the wafer. 

The backside depth profile method 

developed here can be applied to a 

variety of different samples including fully processed, patterned wafers. 

We have prepared three-dimensional chemical images that illustrate artifacts produced during the grinding and polishing procedure. This was accomplished using secondary ion image depth profiling techniques to sequentially collect a 

stack of images during ion sputtering. These images were then combined to produce three-dimensional chemical images. The image below clearly shows the artifact of inclined polishing; the plane of polish is inclined relative to the 

surface of the sample. If not corrected, this artifact will degrade the 

resolution of our SIMS analysis. By applying advanced image analysis 

techniques to the image stack, we minimized the effect of inclined polishing and improved the depth resolution of our SIMS analysis. Future 

plans include combining backside sample preparation with C60+

cluster 

ion SIMS depth profiling to further improve the depth resolution of 

SIMS measurements. 

A CSTL-led research team developed a backside sample preparation method that uses grinding and 

polishing to remove the silicon 

substrate prior to SIMS analysis 

thus improving depth resolution. 

Si: green 

Al: red 

Ti: blue 

90 nm

Si 

ZrAlO 

TiN 

Experimental gate stack 

as profiled 

By improving depth resolution, 

backside SIMS depth profile analysis 

should extend the utility of SIMS 

measurements to the characterization of future-generation semiconductor electronic devices. 
120

Neutron Transmutation Doping of Compound Semiconductors 

R.M. Lindstrom (839) 

Transmutation of silicon by nuclear reactions is used for the production of highpower 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 process is also applicable to compound 

semiconductors. 

The same process is applicable to 1:1 compound semiconductors. Recently, neutron transmutation doping has been explored in two additional systems. In collaboration with the University of North Texas and the NIST Center for Neutron 

Research, films of Hg0.8Cd0.2Te on Cd0.95Zn0.05Te substrates were subjected to neutron 

irradiation. The goal was to transmute both Hg to Au and Te to As. An initial experiment, using neutron fluences of (1 to 2) x1016 cm-2, showed that transmutation is 

feasible. The results of the experiment were 

published in Physica E. To study the effects of 

irradiation quantitatively, additional samples were 

exposed to a pure thermal neutron flux for fluences 

of 3x1016 cm-2 to 1.3x1017 cm-2; this work were reported at the US Workshop on the Physics and Chemistry of II-VI 

Materials held in Chicago during October 2004. The future applications of this technology depend on the results of 

characterization measurements now underway in our partner institutions. 

Properties and Processes for Cryogenic Refrigeration 

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

Details provided in the Industrial and Analytical Instruments and Services Section of this Report 

 

CSTL researcher contributes 

to the investigation of the use 

of neutron transmutation doping for HgCdTe devices. 
121

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 US 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 US national measurement standards. In FY 2004, CSTL performed a total of 682 calibrations for: airspeed, flow, humidity, thermometry, leak, 

vacuum, pressure, volume, and density, serving over 140

customers. In addition, 221 optical filter sets were recertified for wavelength and/or absorbance in the UV and 

NIR spectral regions for more than 136 different companies. 

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 

1400 SRMs; in FY04 NIST sold approximately 30,400 

SRM units to more than 6,500 unique customers. Approximately 24,500 of the units sold were from the ≈1000 

different types of materials that are certified for or support 

measurements of 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 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. 

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 − High-accuracy liquid volume and density, 

hydrocarbon liquid flow 

Humidity − Thermodynamic methods for generation 

of moisture in gases; cavity ring-down spectroscopy 

Chemical Standards Development 

Examples of new reference materials in production 

from CSTL: 

Health − Human cardiac troponin complex, 

homocysteine, folic acid

Dietary Supplements − Ephedra standards, Ginko 

biloba, bitterorange 

Environmental − Contaminants in fish tissue and 

marine sediment, sulfur in diesel fuel, house dust 

Chemicals − Low-carbon steel, zeolites 

Commodities − Aluminum alloys, electronic scrap 

artifact, titanium alloy 

Forensics/Homeland Defense − Trace particulate 

explosives, drugs of abuse, arson test mixture 
122

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 NTRMprogram in gas-mixture reference materials was developed in 1992 for the compressed gas industry. The high availability 

of gas-mixture reference materials as NTRMs has significantly aided vehicle emissions testing in the US and has become the cornerstone of emissions trading in the fossil-fueled electric 

power industry. During FY 2004, 15 new gas NTRMs were certified for 

three companies. These included two batches of very low level, and therefore analytically difficult, 20 μmol/mol nitric oxide in nitrogen NTRMs. 

An additional 29 NTRM batches were recertified this year for 4 companies 

to extend their certification period by 4 years. 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 laborintensive 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 resulted from our 

publication of band positions of dilute acidic holmium oxide solution as an intrinsic wavelength standard. These values 

were determined from results of an international comparison involving fifteen institutions (fourteen National Metrology 

Institutes). 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. 

CSTL scientists continually strive to improve the metrology that underpins our measurements, standards, and data. We 

are also keenly aware that we do not live in isolation in the US – we are part of a global community, with an interdependence that crosses the traditional economic, environmental, and social boundaries. The US-based companies we 

serve are global, and therefore they function and compete in a global market. In order to meet our customers’ growing 

needs we are proactive in the international metrology community to ensure that measurements made in the US are accepted worldwide. Providing the traceability structure for the US promotes equity in trade and industrial competitiveness in the world marketplace. 

Selected Chemical and Physical Standards Activities in FY 2004 

(Described in more detail elsewhere in this report) 

Automotive and Aerospace 

Gas Mixtures Standards for the Automotive Industry: The NTRM Prime Program 

W. D. Dorko (839) 

New Gas Standards for Calibrating Instrumentation Used for Measuring Emissions from Next Generation Low-Emission Vehicles 

W.J. Thorn III (839) 

New Hydrocarbon Liquid Flow Standard for 0.2 L/min to 5 L/min 

T. T. Yeh, P. I. Espina, and J. Aguilera (836) 

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. 
123

Pharmaceuticals and Biomanufacturing 

Leveraging Traceability for Chemical Spectrophotometry Through the Commercial Sector 

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

Energy and Environmental Technologies 

Standards Development and Measurements to Support Global Climate Change 

G.C. Rhoderick (839) 

Standard Reference Materials (SRMs) and Quality Assurance Activities to Support Measurements of 

Organics on Air Particulate Matter Less Than 2.5 μm (PM2.5) 

S.A. Wise, J.R. Kucklick, B.J. Porter, D.L. Poster, M.M Schantz, R.O. Spatz, and R. Zeisler (839) 

Reference Material (RM) 8785 Air Particulate Matter on Filter Media

G.A. Klouda (837), J.J. Filliben (ITL), and H.J. Parish (SRI International) 

New Standard Reference Material (SRM) for Organic Contaminants in House Dust 

M.M. Schantz, J.M. Keller, J.R. Kucklick, D.L. Poster, H.M. Stapleton, S. Vander Pol, 

and S.A. Wise (839) 

Method Development and Measurements of Polybrominated Diphenyl Ethers (PBDEs) in Tissue, Serum, and Sediment Standard Reference Materials (SRMs) 

H.M. Stapleton, J.M. Keller, M.M. Schantz, and S.A. Wise (839) 

Standard Reference Materials (SRMs) for Contaminants in Marine Tissue and Sediment 

S.J. Christopher, R.D. Day, W.C. Davis, S.E. Long, R.S. Pugh, M.M. Schantz, J.R. Kucklick, D.L. Poster, J.M. 

Keller, H.M. Stapleton, K.E. Sharpless, C.S. Phinney, B. J. Porter, E.A. Mackey, R.O. Spatz, C.E. Bryan, J.R. 

Sieber, R. Zeisler, S.A. Wise, and G.C. Turk (839) 

Food and Nutritional Products 

Development of Standard Reference Materials (SRMs) for Dietary Supplements 

S.A. Wise, L.C. Sander, and K.E. Sharpless (839) 

Development of Ephedrine Alkaloid-Based Dietary Supplement Standard Reference Materials (SRMs) 

L.C. Sander, K.E. Sharpless, J. Brown Thomas, B.J. Porter, T.A. Butler, M. Satterfield, S.E. Long, L.A. Mackey, 

K.E. Murphy, L.J. Wood, R.D. Vocke, L.L. Yu, and S.A. Wise (839) 

Forensics and Homeland Security 

Advanced InkJet Printing Technology for Trace Explosives Standards 

G.J. Gillen, R.A. Fletcher, J.R. Verkouteren, M.R. Verkouteren, and G.A. Klouda (837) 

A NIST Reference Material to Support Explosive Device Measurements 

W.A. MacCrehan and M. Bedner (839) 

Candidate Material for an Explosive Residues on Soil Reference Material 

B.A. Benner, Jr. and W.A. MacCrehan (839) 

Ethanol in Water Standard Reference Materials 

M.M. Schantz (839) 
124

Health and Medical Technologies

Technical Procedures for a NIST-Traceable Clinical Reference Laboratory Network 

G.C. Turk, S.E. Long, and D.L. Duewer (839)

Development of Reference Methods and Reference Materials for Clinical Diagnostic Markers 

M.J. Welch, D.M. Bunk, S. S-C. Tai, N. Dodder, B.C. Nelson, M.B. Satterfield, and L.T. Sniegoski (839) 

Reissue of Electrolytes in Human Serum SRM Now Available 

S.E. Long and K.E. Murphy (839) 

Development of Reference Methods and SRMs for Toxic Species in Body Fluids 

S.J. Christopher, W.C. Davis, C.E. Bryan, and R.D. Day (839) 

The Development of Two Standard Reference Materials: Heteroplasmic Mitochondrial DNA Mutation 

Detection Standard (SRM 2394) and Fragile X Human DNA Triplet Repeat Standard (SRM 2399) 

B.C. Levin, D.K. Hancock, and K.L. Richie (831) 

Standard Reference Material for Measuring DNA Damage Related to Disease and Aging 

H. Rodriguez, P. Jaruga, and M.M. Dizdar (831) 

Standards for Fluorescence Microarray Analyses 

G.W. Kramer, P.C. DeRose (839), and A.G. Gaigalas (831) 

Reference Material 8640 for the Calibration of Flow Cytometers 

A. Gaigalas and L. Wang (831) 

Industrial and Analytical Instruments and Services 

Development of a Calibration System for Refrigerant Leaks 

P. J. Abbott (836) 

Critical Reference Materials for Mineral Commodities 

J. R. Sieber and A.F. Marlow (839), S. A. Wilson (USGS), and J. T. Wolsiefer, Sr. (Silica Fume Association) 

Gas Standards Based on Optical Spectroscopies 

J. T. Hodges and P. M. Chu (836), and R. Ciurylo (University of Nicolas Copernicus, Poland) 

New Series of Standard Reference Materials (SRMs) for Raman Intensity Correction 

S. J. Choquette (839), W. Hurst (836), and E. Etz (837) 
125

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 decision-making regarding the health and safety of the US 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 US National Metrology Institute (NMI) and the 

agency of the US Government responsible for US 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 US national 

measurement standards to establish degrees of equivalence of US 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 US standards by US 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. 

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. 

http://kcdb.bipm.org/BIPM-KCDB/

NIST and other National Metrology Institutes around the world have the responsibility for establishing, maintaining, and disseminating the highest level 

of metrological references for a given 

country or economy. The calibration 

and measurement services that these 

NMIs provide must be of high quality 

and delivered to our customers in a consistent and transparent manner. 
126

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 thus, 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 US 

representative 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 (a total of ten), which bring together the 

world's experts in their specified fields as advisers on 

scientific and technical matters. The first of these 

Consultative Committees (Electricity and Magnetism) 

was established in 1927, and the most recent was set 

up in 1998 (Acoutics, Ultrasound and Vibrations). A full listing can be 

found on the BIPM website. 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. 

The Consultative Committee for Temperature (CCT) was set up in 1937. 

Present activities concern matters related to the establishment and realization of the International Temperature Scale of 1990 (ITS-90) and thermodynamic temperature, extension and improvement of the ITS-90, secondary reference points, and international reference tables for thermocouples 

and resistance thermometers. NIST leads or is a member of eight of the 

nine Working Groups. CSTL staff members chair three of the Working 

Groups (Defining fixed points, Humidity measurements, and Calibration 

and measurement capabilities) and are members of three other Working 

Groups (Key comparisons, Uncertainties, and Thermodynamic temperature determinations). 

The Consultative Committee for Mass and Related Quantities (CCM) was established in 1980. There are eleven Working Groups and present activities concern matters related to the comparisons of mass standards with the international 

prototype of the kilogram, considerations affecting the definition of the unit of mass, also density, pressure and force 

standards. CSTL interests overlap with five of the Working Groups (High Pressure, Low Pressure, Medium Pressure, 

Fluid Flow, and Viscosity) and a total of seven staff members participate in these Working Groups, with two serving as 

chair (although one has retired this year). 

Consultative Committee for Amount of Substance: Metrology in Chemistry (CCQM) was created in 1993. Present activities concern primary methods for measuring amount of substance, and international comparisons, establishment of 

international equivalence between national laboratories, and advice to the CIPM on matters concerned with metrology 

in chemistry. CSTL is active in all seven of the CCQM Working Groups: Gas Analysis; Organic Analysis; Inorganic 

Analysis; Electrochemistry; Bioanalysis, Surface Analysis; and, 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. The CSTL director chairs 

the Organic Analysis Working Group, and a CSTL division chief co-chairs the Bioanalysis Working Group. 

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 key comparisons 

(KCs). During the past three years, eight intercomparison exercises were carried out to assess the proficiency of SIM NMIs and/or their 

designated laboratories. Five additional exercises are planned for 2005. 

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 
127

Participation in CCQM Key Comparisons is available only to top-tier NMIs around the world. Within the Americas, 

only the US, Canada, and Mexico have well-established programs in chemical metrology. While SIM is focusing entirely on training and capability assessment, Regional Chemical Metrology Working Groups in Europe and the Asian 

Pacific are forging ahead and conducting MRA-driven Key Comparison Studies. We have established agreements with 

the Chemical Metrology Working Group Leaders of both regions to allow non-CCQM member countries within SIM to 

participate in such studies as soon as they feel competent to do so. Once self-assessed capabilities of SIM member 

states are at an appropriate level, as determined based on performance in the SIM capability assessment studies, they 

can also request permission to participate in CCQM Comparisons. 

Quality Systems Support Calibration and Measurement 

Capabilities (CMC) claims: A listing of Calibration and 

Measurement Capabilities is Appendix C of the BIPM Key 

Comparisons that was constructed as a means to implement 

the CIPM Mutual Recognition Arrangement. It is also required that a quality system be in place to support CMC 

claims. The NIST-level quality manual has been adopted for 

use and conforms to the requirements of ISO 17025. Each 

NIST Laboratory/Division disseminating measurement 

standards is required to have a fully documented quality 

system in place that summarizes and formalizes policies and 

approaches for addressing quality-related issues concerning 

the services that it provides. The approach is uniform across 

NIST, multi-tiered and modular. This quality system is used 

to support CMC claims both for chemical and physical 

measurement services provided by CSTL. 

Selected Key and International Comparisons Completed in FY 2004 

International Comparisons in Electrochemical Analysis 

K.W. Pratt (839) 

NIST participated in three international comparisons in the area of electrochemical analysis: SIM.8.11P in pH measurement, CCQM-K34 in assay of potassium hydrogen phthalate (KHP), and CCQM-P47 in electrolytic conductivity. 

SIM Pilot Study SIM.8.11P, piloted by CENAM, was a follow-up to the previous 

SIM.8.P4, which had been piloted by NIST in 2000. Five participants, including 

NIST, performed Harned cell pH methods (primary method). The NIST results 

were in excellent agreement (within ±0.0017 pH) with other experienced NMIs 

(those that had participated in previous CCQM pH Key Comparisons CCQM-K9 or 

CCQM-K17). The NIST uncertainties, which included all known Type A and Type 

B sources, were equal to or smaller than those of the other participating NMIs in the 

primary measurement. The reduction in the NIST combined uncertainty resulted 

from a threefold reduction in the Type A uncertainty of the extrapolation of the acidity function to obtain pa°, the acidity function in buffer without added chloride 

(from which the pH is directly obtained). This reduction was directly attributable to 

the implementation of pre-equilibration of the Ag|AgCl electrodes used in the 

Harned cells. 

CCQM Key Comparison CCQMK34 was designed to evaluate the 

agreement obtainable for the assay of potassium hydrogen phthalate 

(KHP) using high-accuracy assays. The seven participants each used 

coulometry. The NIST result and its estimate of uncertainty agreed well 

Chemical Metrology: About 1000 of the 3000 

CMCs for chemical measurements in the 

BIPM database are from NIST/CSTL. 

CSTL has participated in 80 of the 93 Key 

Comparisons in chemical metrology and has 

led 40. 

Physical Metrology: 

CSTL also has CMCs for Flow (14), Temperature and Humidity (99), Vacuum and 

Pressure (23), and is participating in or leading approximately 10 KCs in any given year. 

NIST is the pilot lab for KC Low-Pressure 

Gas Flow, the transfer standard development is near completion and the comparison 

is scheduled for mid FY05. 

Harned Cell

The results of this Pilot Study correspond directly to the procedure for 

the certification of the phosphate pH 

SRM 186g and support the validity of 

its certification. 
128

with those of the other “experienced” NMIs (who had participated in the corresponding pilot study, CCQM-P36). In 

addition, the NIST estimate of uncertainty was more complete than those submitted by all but one of the other participants (one included the same set of sources), in that more possible sources of uncertainty were included in the estimate. 

CCQM Pilot Study CCQM-P47 evaluated the performance of 

NMIs in the measurement of electrolytic conductivity of two 

KCl solutions of nominal conductivity equal to 50 mS/m (500 

μS/cm) and 5 mS/m (50 μS/cm). For the 5 

mS/m solution, the pilot laboratory (NMi, Netherlands) discovered a time-instability in the solution as stored in the bottles used for distribution 

of the solution. Following discussion of the 

original results among the participants in April 2004, the pilot 

laboratory, NMi (Netherlands), decided to institute a timedependent correction of the reference value. For the 50 mS/m 

solution, the NIST result was initially higher than the reference 

value. Following discussion at the CCQM Electrochemical 

Analysis Working Group, April 2004, the cell calibration procedure used at NIST was modified to eliminate the non-IUPAC 

primary calibrants that previously had been used. With this 

modification, the NIST result for the 50 mS/m solution was in 

excellent agreement with the reference value. 

Sulfur in Diesel Fuel by Isotope Dilution Mass Spectrometry: Results of the CCQM K35 Key 

Comparison 

W.R. Kelly, R.D. Vocke, J.L. Mann, and G.C. Turk (839) 

Regulations in North America and Europe require that the petroleum industry and regulatory environmental agencies be 

able to measure accurately sulfur in diesel fuel between 5 μg/g and 50 μg/g with a total combined uncertainty of less 

than 1 μg/g. Both Europe and the United States 

will be moving to sulfur limits of 50 μg/g and below fuels in the next year. Lower sulfur in fuels 

will make possible extremely efficient and longlived after-treatment technologies based on noble 

metal catalysts. 

NIST planned and conducted a CCQM Key Comparison (K35) and a concurrent pilot study (P26.1) 

on sulfur in diesel fuel and kerosene, and the results were reported to the Inorganic Working 

Group of the CCQM in April 2004. 

The four laboratories shown in red were registered 

Key Comparison participants using isotope dilution mass spectrometry either with thermal ionization instruments (NIST and BAM) or inductivelycoupled plasma mass spectrometry (LGC and 

IRMM). The other values were submitted as Pilot 

Study results. Shown in the figure are the mean 

values and total expanded uncertainties expressed 

as 95% confidence intervals and the individual 

determinations (solid circles to the right of the 

In addition to the above three comparisons, 

NIST also completed and submitted to the 

CCQM the Final Report of Pilot Studies 

CCQM-P19/P19.1, Assay of 0.01 mol kg-1

Hydrochloric Acid, in which NIST was the 

pilot laboratory. The analyses for the two 

phases of this Pilot Study had been completed in preceding years, with 14 participants from NMIs worldwide. The submission of the Final Report formally completes 

this study and constitutes the official record 

of its results. 

Participants in the studies measured 

sulfur concentrations in three candidate SRMs covering a wide range of 

sulfur concentrations. 

1234567

r

38

39

40

41

42

43

44

NIST

BAM

LGC IDMS IRMM

BAM

UVF

CENAM

XRF

NIST

XRF

Key Comparison Reference Value computed using Labs 1-4.

KCRV (42.17)

U

U

NIST led a Key Comparison and associated Pilot 

Study to assess and document the capability of 

National Metrology Institutes (NMIs) to perform 

accurate determinations of sulfur in diesel fuel at 

and below future regulatory limits. 

SRM 2770 

Reported sulfur concentrations in ug/g by 

seven laboratories including NIST. The Key

Comparison Reference Value is 42.17 μg/g. 
129

estimates) submitted by the NMIs. The Key Comparison Reference Value (KCRV) was computed using the Mixture 

Model Median method developed at NIST. The agreement among the four NMIs is considered good since the computed 

standard deviation is less than 1 μg/g. 

The results on all three samples are shown in the figure 

to the right as deviations from the reference values in 

relative percent. The reference values and total uncertainty are indicated by the points and error bars that are 

centered on the zero reference line. The reference values have been arbitrarily shifted to higher values for 

clarity of presentation. The greater spread in the data at 

the lower concentrations is a consequence of the greater 

influence of the blank. 

CCQM K35 Key Comparison has demonstrated the capability of 4 NMIs to perform sulfur measurements in 

diesel fuel at a nominal 40 μg/g concentration. Today 

the regulated upper sulfur limit in US on-road diesel fuel 

is 500 μg/g, but this limit will drop to 15 μg/g in year 

2006 and perhaps to near-zero (< 5 μg/g) in the next decade. Japan and Europe will adopt a 50 μg/g limit in 

years 2004 and 2005. Europe will phase in a 10 μg/g 

limit between 2005 and 2009 and Japan will adopt the 

same limit in 2007. The incremental measurement challenges increase non-linearly as sulfur concentrations decrease from 500 μg/g to near zero levels. The regulatory 

agencies and the petroleum industry look to the national 

metrology laboratories to produce calibration standards 

with certified concentrations and uncertainties that will 

ensure a smooth and cost effective transition to low sulfur diesel fuel. CCQM K35 and P26.1 address present 

and near-future measurement needs at the NMI level. 

The Joint Committee on Traceability in Laboratory Medicine (JCTLM) 

Identification and Provision of “Higher Order” Certified Reference Materials and Reference 

Measurement Procedures Required for US Industry Compliance with the EU IVD Directive 

W.E. May (839) and V.L. Vilker (831)

The goal of obtaining comparability of laboratory diagnostic test results will be possible only when common 

reference systems can be established for worldwide use. 

A critical step in reaching this goal is achieving traceability of reference measurement procedures and reference 

materials to a universally recognized and accepted reference point such as the International System of Units (SI). 

Recently, traceability requirements for medical devices to 

be imported into the European Community have been 

codified. The European Community In Vitro Diagnostic 

Directive (EC IVDD) states that “The traceability of values assigned to calibrators and/or control materials must 

be assured through available reference measurement procedures and/or available reference materials of a higher 

order” (98/79/EC, Annex1 (A) (3) 2nd paragraph). The 

Joint Committee on Traceability in Laboratory Medicine 

Sulfur μg/g

1 10 100 1000 10000

Deviations of IDMS Means Relative to KCRV or RV

-8

-6

-4

-2

0

2

4

6

8

BAM

LGC

Deviations

in % Relative

IRMM

NIST

“Worldwide, many jurisdictions have recognized 

the public health and environmental costs of allowing motor vehicle fuels to contain High levels of sulfur. The EU, US, and Japan have led the way in 

sulfur reduction, and will reach near-zero sulfur 

levels later in this decade.” 

The International Council on Clean Transportation

The US IVD industry (ADVAMED) has asked 

NIST to work with our counterparts in Europe 

and the Asia-Pacific to provide the reference 

materials and methods of “higher order” that 

are urgently needed to comply with the requirements of the EU IVD Directive. Without 

timely completion of these standards, the industry’s access into the European market is seriously jeopardized. 
130

(JCTLM) was created to meet the need for a worldwide platform to promote and give guidance on internationally recognized and accepted equivalence of measurements in laboratory medicine and traceability to appropriate measurement 

standards. At present, neither reference materials nor reference methods are available for the vast majority of the 

chemical or biochemical species that are measured in medical laboratories using IVDs on a routine basis. 

NIST has provided the clinical measurements community with both neat chemical and human body fluid-based Standard Reference Materials for well-defined health status markers such as eElectrolytes (sodium, potassium, lithium and 

magnesium), cholesterol, creatinine, glucose, triglycerides, urea, uric acid, vitamins A, C, E and beta carotene, and several therapeutic and drugs of abuse for more than 20 years. Over the past few years, NIST has expanded its standards 

program in clinical diagnostics. A major portion of the expansion of the 

NIST program has involved the development of new standards for several 

protein, hormone, peptide, and other large biomolecule-based health 

status markers. Efforts have focused on development of reference methods and SRMs for: cardiac troponin I (heart attack occurrence and damage), C-reactive protein and homocysteine (heart attack risk), cortisol (endocrine function), folates (neural tube defects), 

glycated hemoglobin (diabetes status), prostate specific antigen (prostate cancer), and triiodothryonine and thyroxine 

(thyroid function). Many of these new markers show great 

promise from the clinical diagnostic perspective, but offer 

new and more difficult challenges for standardization. They 

are present in the blood at very low concentrations and many 

are thermally labile, very polar, and heterogeneous − both in 

conformation and in what is attached to them. Because of the 

vast market for tests for these new markers, many different 

measurement approaches have been developed that often 

provide quite different results. These discrepancies can lead 

to erroneous diagnoses and/or the need for retesting − both of 

which are very costly. 

NIST led the efforts of the JCTLM Working Group on Reference Materials and Reference Laboratory Procedures in 

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 EU 

IVD Directive regarding in vitro diagnostic medical devices. In order to facilitate a fair and transparent review process, 

eight analyte categories were identified and review teams established for each. To the extent possible, each review team 

has representation from IVD manufacturers, National Metrology Institutes, accreditation organizations, and professional 

societies from the US, Europe, and the Asia Pacific Region. 

On April 1, 2004, the JCTLM published its first List of Higher Order Reference Materials and Reference Measurement 

Procedures. This initial list (List I) comprises Certified Reference Materials (CRMs) and Reference Measurement Procedures for well-defined chemical entities or internationally recognized reference method-defined measurands (see examples in tables on the following page). Reference materials and measurement procedures included in List I are those 

that provide values that are traceable to the SI units; e.g., electrolytes, enzymes, drugs, metabolites and substrates, nonpeptide hormones, and some proteins. List II was published during the last quarter of 2004, and comprises International 

Conventional Reference Materials, i.e., those for which the measurand(s) is/are not SI-traceable and/or no internationally recognized 

reference measurement procedure is available; for example, the 

WHO reference materials for coagulation factors, nucleic acids, and 

some proteins. 

The current list contains approximately 100 reference measurement procedure entries for 58 different health status 

markers. Thirty of these “higher order” reference measurement procedures are from NIST. For reference materials, the 

list contains approximately 150 entries for 96 measurands; NIST SRMs provide traceability for 72 of these. A laboratory-based quality assurance audit program has been initiated to provide measurement results regarding the comparabilThe JCTLM was established by the International 

Committee of Weights and Measures (CIPM), International Federation of Clinical Chemistry (IFCC) 

and Laboratory Medicine, and International Laboratory Accreditation Cooperation (ILAC) 

NIST expands its clinical reference 

material program, and develops new 

reference methods. 

Availability of NIST Clinical Standards: 

Three new SRMs were completed: 

• Cardiac Troponin I (SRM 2921) 

• Toxic Elements in Urine (SRM 2670a) 

• Homocysteine and Folate in Human Serum 

(SRM 1955). 

Three high-priority SRMs were renewed: 

• Electrolytes in Human Serum (SRM 956b) 

• Glucose (SRM 965a) 

• Lipids in Human Serum (SRM 1851b). 
131

ity of multiple “higher order” reference materials for the same measurand on the list as well as to verify the veracity of 

the review process. Nominations are currently being accepted for high-purity substance reference materials and for human hair and body fluid-based reference materials and reference measurement procedures in the eight original plus five 

new analyte categories. 
132
133

12. Data and Informatics 

... assuring that US Industry has access to accurate and reliable data 

 and predictive models 

For 30 years, NIST has provided well-documented numeric data to scientists and 

engineers for use in technical problem-solving, research, and development. These 

recommended values are based on data that have been 

extracted from the world's literature, assessed for 

reliability, and then evaluated to select the preferred values. These data activities 

are conducted by scientists at NIST and in university data centers. 

One of CSTL’s goals is to assure that US 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 80 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. 

One data area of increasing focus for CSTL is bioinformatics. CSTL researchers work to develop adaptive, automated 

methods of processing and presenting biological and chemical data using connection tables that are sufficiently flexible 

and easy to use and allow users to find, with confidence, information for the most structurally-relevant data used in 

structure-based drug design. This year NIST, in collaboration with 

NIH-NCI, unveiled an online database that contains the structures of 

HIV protease and compounds targeted against this enzyme. This database permits faster and more reliable access to standardized data related 

to the design and development of compounds against HIV. It will also 

provide improved resources for analyzing drug resistance to medications that are currently used to treat AIDS. The availability of such a 

resource to industry is expected to foster the development of new and 

better drug products. More details are provided in the Health and 

Medical Technologies section of this report. CSTL scientists also maintain other web-based bio-related databases: the 

Human Mitochondrial Protein Database and the Enzyme Thermodynamics Database. 

NIST researchers are working with other scientists and organizations to establish data standards and more rapid methods of data entry in a number of data areas, including structural biology, thermodynamics, and kinetics. One example is 

the Thermodynamics Research Center (TRC). The TRC group 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?” In 2004, ThermoML was completed with 

incorporation of extensions for critically evaluated data, predicted data, and equation representations. It was also accepted as the foundation for the development of the IUPAC (International Union 

of Pure and Applied Chemistry) standard for thermodynamic data communications. In addition, in order to build an 

infrastructure for the process of global thermodynamic data communication, Guided Data Capture (GDC) software was 

CSTL continues to be a 

prominent source of SRD 

products at NIST. In FY 

2004, 3807 Mass Spectral 

units were sold through distributors to the GC/MS 

community, 363 Thermodynamics Research Center publications were distributed, and 654 units of CSTL 

databases were distributed 

directly to customers. In addition, over 2,500 CSTL 

products such as dynamic 

link libraries (DLLs), fluid 

property coefficients, and 

source code portions were 

incorporated into commercial 

software packages. 

NIST in collaboration with NIH developed a new structural database 

for AIDS research using novel techniques to annotate and browse. 

http://xpdb.nist.gov/hivsdb/hivsdb.html 

IUPAC 
134

WebBook Statistics: 

• over 650,000 distinct IP addresses access 

the WebBook each year

• between 10K and 20K users per week with 

around 50% return users

• an estimated average increase of about 

28% in usage of the WebBook 

http://webbook.nist.gov/chemistry 

developed for mass-scale abstraction of experimental data from the literature. Additional TRC details are provided later 

in this section. 

Over 2,500 NIST Mass Spectral Libraries are installed on GC/MS instruments each year. The most recent version of 

the library was released in 2002, and it remains the most comprehensive, reliable library of mass spectral ‘fingerprints’ 

to assist in the task of compound identification by GC/MS. GC/MS is the most widely used analytical tool for low concentration analysis for food safety and environmental monitoring. In addition it is extensively used in general organic 

analysis, the development of new flavoring agents, the analysis of fragrances and in many medical applications. However, at extremely low concentrations, it can be difficult to extract the trace signal from the mass spectrum due to the 

very complex background that is present. Therefore, a deconvolution software tool was developed by NIST. The 

Automatic Mass spectral Deconvolution and Identification Software (AMDIS) was originally developed for detection 

of chemical weapons in complex mixtures such as might be found in the environment or in chemical process streams. It 

was designed to work without analyst input as a method of insuring that sensitive business information that could be 

present in a process stream was not compromised. In the last year the growth in the use of AMDIS by the organic analytical community has been very strong. One of the most exciting developments has been the incorporation of AMDIS 

into a new set of tools for automatic analysis developed by Agilent Technologies. The tools have been given the general name of Deconvolution Reporting Software (DRS) and incorporate Agilent 

Technologies run-time locking technology, the NIST search software, and AMDIS 

in a combined tool to allow users to identify pesticides at lower concentrations and 

with more confidence than had been possible with the Agilent system alone. More details are provided in the article 

entitled: AMDIS – Automatic Mass Spectral Deconvolution and Identification Software in the Industrial and Analytical Instruments section of this report.

The NIST Chemistry WebBook remains one of the most used resources for chemical and physical property data. The 

numbers of users, between 10,000 and 20,000 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 45% to 55%, is a good 

indication that the user community feels that the resource is valuable. The NIST Chemistry WebBook has been awarded 

"Best Chemistry Site on the Web - Portals and Information Hubs" by ChemIndustry.com Inc., John Wiley and Sons, 

Inc., and the Royal Society of Chemistry, UK. The WebBook is 

second in total use among chemistry database web sites (only the 

Chemical Abstracts site has higher usage) and over 2,500 sites 

directly link to the WebBook, including essentially every 

technical library in the world. This year the Chemistry WebBook 

has been made available in other language versions; see details in 

The NIST Chemistry WebBook Goes Multilingual in the 

section that follows. 

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, in particular those working on IUPAC and ASTM committees. 

Selected Data Activities for FY 2004 

The NIST Chemistry WebBook Goes Multilingual 

P. Linstrom and W. G. Mallard (838) 

The role of international trade is growing more important in the US 

economy each year. Although most US goods have a natural market in 

the Western Hemisphere, their export continues to be hampered by numerous factors. One such factor has been our limited ability to communicate with the non-English-speaking world. To address this challenge, 

NIST is actively working to make more of its information available in 

Spanish. The WebBook translations are part of this effort. 

The NIST Chemistry WebBook is 

a widely used tool for both teaching and practicing chemistry 

worldwide, therefore NIST has 

made the WebBook available in 

more languages. 
135

The need for as much information to be available in as many European languages as possible has been a driving force 

for numerous EU projects. Therefore, we have been cooperating with the European Commission to make the WebBook 

available in more languages. Since the NIST WebBook is extensively used in Europe this made it a prime candidate for 

the EU effort. The NIST role has been to provide the basic text in marked-up format ready for translation. The cost of 

the translation has been born by the EU. The first results of this are a set of web pages allowing the basic search to be 

done in French, Spanish, Czech, and Portuguese. 

The W3C (World Wide Web Consortium) 

set standards for multilingual sites and they 

are supported by most of the new browsers. 

Using these standards, a Spanish-speaking 

user of the WebBook would be greeted in 

Spanish, and a user from Brazil would be 

greeted in Portuguese. Examples are shown 

to the right. In the future other languages 

will be added as the EU provides additional 

translation. 

While it is impractical to translate every 

section of the WebBook, there are major portions of the opening screens that allow the users in other languages to more 

readily use the complex search features of the WebBook. We sought the expertise of Rui Pinto from Departamento de 

Quimica, Universidade de Aveiro, Portugal who came to NIST as a Guest Researcher to assist in this project, primarily 

to complete the Portuguese, Spanish, and French translations. 

Because the WebBook has been conforming in its design to the most rigorous standards of W3C, the use of multiple 

languages with full support of the language-specific character sets was relatively straightforward to implement. 

This meant that the preceding English version of the “disclaimer” could become what follows for the Czech version. 

Future changes will only require that the text be made available in standard formatted files. 

Software for Phyloinformatics: Nextool, Nexplot and a NEXUS API in Perl 

A. Stoltzfus (831), C. Liang, P.J. Yang (CARB/UMBI), W. Qiu and B. O'Brien (Hunter College, CUNY, NY) 

Biological data make more sense when analyzed comparatively, in a historical (evolutionary) context, though it is 

often technically difficult to do so. Genome annotation, 

drug target discovery, biomolecule engineering, and medical genetics have come to rely increasingly on relatively 

crude forms of comparative sequence analysis. The success 

of a more robust approach will depend on software tools 

that facilitate the storage, exchange, processing, and visualization of genomic data together with phylogenetic trees. Our 

work focuses on developing and applying "phyloinformatics" tools that combine the rigor of evolutionary analysis with 

the scope of bioinformatics. These tools include procedures for data analysis, software applications, and database systems. 

The National Institute of Standards and Technology (NIST) uses its best efforts to deliver a high quality 

copy of the Database and to verify that the data contained therein have been selected on the basis of sound 

scientific judgment. However, NIST makes no warranties to that effect, and NIST shall not be liable for any

damage that may result from errors or omissions in the Database. 

Národní Institut pro standardy a technologii (NIST) vynakládá maximální úsilí, aby zpřístupnil vysoce 

kvalitní databázi a mohl potvrdit, že zde obsažená data byla vybrána na základě řádného vědeckého posouzení. Přesto však NIST v této věci nedává žádné záruky a NIST nebude zodpovědný za jakoukoliv 

škodu, která může vzniknout v důsledku chyb nebo opominutí v Databázi. 

The NIST/UMBI/CUNY research team is developing procedures, software tools, and databases for analysis of genomic data that combine the rigor of evolutionary analysis with the 

scope of bioinformatics. 

Libro del Web de Química del NIST 

Base de Datos de Referencia Estándar del NIST Número 69 

Publicada en Noviembre, 2004 

NIST Livro de Química na Web 

Base de dados de Referência padrão do NIST número 69 

Publicada em Novembro, 2004
136

For over a decade, evolution researchers 

have relied successfully on a little-known 

data exchange format for comparative 

analysis known as NEXUS, which stores 

data sets, such as sequences to be compared, together with trees. To adapt this 

format and render it more accessible to the 

genome analysis industry, we have developed a NEXUS Applications Programming 

Interface (API) in Perl, the most commonly 

used computer language in bioinformatics. 

The utility of this software tool-box is 

demonstrated by two applications, Nextool 

and Nexplot. Nextool is a scriptable editor 

designed to automate tasks such as extracting subsets of data from a NEXUS file. 

Nexplot is a visualization tool that produces a customizable PostScript plot that 

combines sequence data (or other data) 

with a tree as shown in the figure. 

ThermoML – An Emerging IUPAC Standard for Thermodynamic Data Communications 

M. Frenkel, R. D. Chirico, Q. Dong, G. R. Hardin (838), and V. V. Diky (Belarussian State University) 

Establishing efficient means for thermodynamic data communications is absolutely critical for provision of solutions to such technological challenges as elimination of data processing redundancies and data collection process duplication, creation of comprehensive data storage facilities, and rapid data propagation from the 

measurement to the data management system and from the data 

management system to engineering applications. Taking into account the diversity of thermodynamic data and numerous methods 

of their reporting and presentation, standardization of thermodynamic data communications is very complex. The 

ThermoML structure represents a balanced combination of hierarchical and relational elements. The ThermoML 

schema structure explicitly incorporates structural elements related to basic principles of phenomenological thermodynamics: thermochemical and thermophysical properties (equilibrium and transport), state variables, system constraints, 

phases, and units. Meta- and numerical data records are grouped into ‘nested blocks’ of information corresponding to 

data sets. The structural features of the ThermoML metadata records ensure unambiguous interpretation of numerical 

data and allow data-quality control based on the Gibbs Phase Rule. ThermoML covers essentially all experimentally 

determined thermodynamic and transport property data (more than 120 properties) for pure compounds, multicomponent mixtures, and chemical reactions (including change-of-state and equilibrium). 

Definitions and descriptions of all quantities related to the expression of uncertainty in ThermoML conform to the Guide to the Expression of Uncertainty in Measurement (ISO, 1993). In 

order to implement this extension of ThermoML, the TRC Group (formerly the Thermodynamic 

Research Center) made an interpretation of the US Guide to the Expression of Uncertainty in 

Measurement for the field of thermodynamics. In 2004, ThermoML was completed with incorporation of extensions for critically evaluated data, predicted data, and equation representations. In early 2004, ThermoML 

was accepted as the foundation for the development of the IUPAC (International Union of Pure and Applied Chemistry) 

standard for thermodynamic data communications. 

In order to build an infrastructure for the process of global thermodynamic data communication, 

Guided Data Capture (GDC) software was developed for mass-scale abstraction from the literature 

of experimental thermophysical and thermochemical property data for organic chemical systems 

involving one, two, and three components, chemical reactions, and chemical equilibria. 

Researchers in the TRC Group have 

developed ThermoML, an XML (Extensible Markup Language)-based approach for storage and exchange of 

thermophysical and thermochemical 

property data. 

GDC
137

Combination of the software 

tools incorporating GDC and 

ThermoML allowed establishment of a new data communication process, which 

now includes major journals 

in the field of thermodynamics, such as the Journal of 

Chemical and Engineering 

Data, The Journal of Chemical Thermodynamics, Thermochimica Acta, and Fluid 

Phase Equilibria. As a result 

of the implementation of this 

process, hundreds of authors 

worldwide generate ThermoML files of their reported 

data at the time of publication. The TRC Group has 

designed and now supports 

Web distribution of the 

ThermoML files in the public 

domain without restriction. 

The TRC Group has also been working with major data-user organizations (Aspentech, US; National Engineering Laboratory, UK; Fiz Chemie, Germany; Virtual Materials Group, Canada; Korean Institute of Science and Technology Information) to develop software ‘readers’ of the ThermoML files. In the near 

future ThermoML will be expanded to provide compound identification using 

the IUPAC-NIST Chemical Identifier (INChI). 

ThermoData Engine: New Generation Expert System for Thermodynamic Data Critical Evaluation 

M. Frenkel, R. D. Chirico, Q. Dong, C. Muzny, (838), V. V. Diky (Belarussian State University), and 

X. Yan (Texas A&M) 

Traditionally, critical data evaluation is an extremely time- and resourceconsuming process, which includes extensive use of labor in data collection, data mining, analysis, fitting, etc. Because of this, it must be performed far in advance of a need within an industrial or scientific application. In addition, it is quite common that by the time the critical dataevaluation process for particular chemical system or property group is 

complete (sometimes after years of data evaluation projects involving 

highly skilled data experts), it must be reinitiated because significant new 

data have become available. This type of slow and inflexible critical data 

evaluation can be defined as ‘static.’ These shortcomings have become 

magnified dramatically within the last 5 to 10 years due to the significant 

increase in the rate of publication of experimental and predicted thermodynamic data to be analyzed during the critical data evaluation process. 

To address the weaknesses of “static” evaluations, the concept of a dynamic data evaluation system was developed at 

NIST. This concept requires very large electronic databases capable of storing essentially all experimental data known 

to date with detailed descriptions of relevant metadata and uncertainties. The combination of these electronic databases 

with expert-system software, designed to automatically generate recommended data based on available experimental 

data, leads to the ability to produce critically evaluated data dynamically or “to order”. The dynamic data evaluation 

process dramatically reduces the effort and costs associated with anticipating future needs and keeping static evaluations 

current. 

The NIST TRC Group developed 

the concept of a dynamic data 

evaluation. It combines electronic 

databases with expert-system software (designed to automatically 

generate recommended data based 

on available experimental data) to 

produce critically evaluated data ‘to 

order’. 

Measurements

J. Chem. Eng. Data

(ACS)

J. Chem. Thermodyn.

(Elsevier)

Fluid Phase Equilibria

(Elsevier)

Int. J. Thermophys.

(Kluwer)

Thermochimica Acta

(Elsevier) Industry: DIPPR

IUPAC

International Association of Chemical 

Thermodynamics (IACT)

Committee on Printed & 

Electronic Publication (CPEP)

NIST/TRC

WEB

Thermodynamicists

Applications

AspenTech

(USA)

Virtual Materials Grp.

(Canada)

Nat’l Engineering 

Lab (UK)

Guided Data

Capture (GDC)

“Reader”

Software

Industrial Engineers

Global Data Communication Process

Fiz Chemie

(Germany)

Measurements

J. Chem. Eng. Data

(ACS)

J. Chem. Thermodyn.

(Elsevier)

Fluid Phase Equilibria

(Elsevier)

Int. J. Thermophys.

(Kluwer)

Thermochimica Acta

(Elsevier) Industry: DIPPR

IUPAC

International Association of Chemical 

Thermodynamics (IACT)

Committee on Printed & 

Electronic Publication (CPEP)

NIST/TRC

WEB

Thermodynamicists

Applications

AspenTech

(USA)

Virtual Materials Grp.

(Canada)

Nat’l Engineering 

Lab (UK)

Guided Data

Capture (GDC)

“Reader”

Software

Industrial Engineers

Global Data Communication Process

Fiz Chemie

(Germany)

Recommendations to 

IUPAC for a formal approval of ThermoML as a 

new IUPAC standard will 

be finalized in FY 2005. 
138

Functional comparison of static and

dynamic data evaluation concepts. 

Critically evaluated data produced by the deployment of the dynamic data-evaluation concept 

can rigorously be characterized with their quality 

assessments providing the ability to propagate 

reliable data-quality limits to all aspects of 

chemical process design. Implementation of the 

dynamic data evaluation concept consists of the 

solution of a number of major tasks: (1) design 

and development of a comprehensive database 

system structure based on the principles of physical chemistry and capable of supporting a largescale data entry operation for the complete set of 

thermophysical, thermochemical, and transport 

properties for chemical systems including pure 

compounds, binary mixtures, ternary mixtures, 

and chemical reactions; (2) development of software tools for automation of the data-entry process with robust and internally-consistent mechanisms for automatic assessments of data uncertainty; (3) design and development of algorithms 

and software tools to assure quality control at all 

stages of data entry and analysis; (4) development 

of algorithms and computer codes to implement 

the stages of the dynamic data-evaluation concept; (5) development of algorithms to implement, target, and apply prediction methods depending on the nature of the chemical system and 

property, including automatic chemical structure 

recognition mechanisms; and (6) development of 

procedures allowing generation of output in a 

format suitable for application in major commercial simulation engines for chemical-process design. 

The ThermoData Engine (TDE) software incorporates all major stages of the concept implementation including data 

retrieval, grouping, normalization, sorting, consistency enforcement, fitting, and prediction. The SOURCE data system 

is used in conjunction with TDE as the comprehensive storage facility for experimental thermophysical and thermochemical property data. In addition the NIST/TRC Ideal Gas Database is used as a source of thermodynamic property 

data in the ideal-gas state. 

In FY 2004, the ‘beta’-version of the first release of the TDE, focused on the thermodynamic properties of pure compounds, was produced and extensively tested within and outside NIST. 

The software architecture emphasizes enforcement of consistency between related properties (including those obtained from predictions), 

assumes an imperfect source of original data, provides for flexibility in 

selection of default data models depending on the 

particular data scenario, incorporates a large variety of models for secondary fitting, and allows 

saving of critically evaluated data in the ThermoML format. The latter assures compatibility of 

the TDE software with any engineering application equipped with a 

ThermoML software ‘reader.’ 

The “alpha” version of the TDE is scheduled for public release in FY05. Further 

development will include incorporation of 

the computational tools for generating 

equations of state on-demand depending 

on the data “scenario.” Longer-term plans 

include expansion of the TDE to critical 

data evaluation for binary mixtures. 
139

The IUPAC NIST Chemical Identifier (INChI) 

D. Tchekhovskoi, S. Stein (838), S. Heller (Department of Agriculture, retired) 

The question of clearly identifying a chemical has been present 

almost since the beginnings of modern chemistry. As the number of chemicals grew, the need for systematic naming produced a number of results. The most widely adopted of these is 

that of the International Union of Pure and Applied Chemistry 

(IUPAC). But for many chemicals the resulting names are complex and so common names are still widely used. For most 

chemists, the graphical structure is the best method for identifying a chemical. The structure provides graphic information that 

can allow a rapid understanding of the properties of the chemical that a long text name can never provide. 

The complications of names even for very simple molecules are 

illustrated below: 

O

NH2

O

OH

NH2

O

2-amino-4-cyclohexene-1,3-dione 6-amino-5-hydroxy-2-cyclohexen-1-one 

The numbering of both the amino group and the double bond changes in these names because of a set of rules for establishing precedence in functional groups. The structures allow the chemist to see the direct connection between the two 

molecules. 

The complication with structures is that they are not easy to order in the 

sense that names can be ordered by simple alphabetical rules and they do 

not have an obvious storage method in the computer. Thus there has 

been a need to develop methods of describing a structure with a simple 

string that can be used both to order sets and to find a specific compound. Computer storage methods and search algorithms have been able 

for some time to process structural data but these have not been standardized (there is no agreed upon alphabet for structure representation in 

computers) and so the various systems for processing the data have not 

been able to easily communicate. 

To accomplish this, much of what is normally viewed as “chemical information” was discarded and the molecules were trimmed to the minimum information needed to differentiate one from the other. In addition, 

a layered approach was developed to deal with some of the more complex issues of chemical structure. 

For example the two molecules in the illustration 

differ only in that one has the two methyl groups 

on the same side relative to the plane of the ring 

(on the right – called cis) and the other has the two 

methyl groups on opposite sides of the ring (in the 

center – called trans). On the left is a diagram that 

can be used to represent either of the molecules. 

The left diagram shows only the connectivity and 

does not specify if the molecule is cis or trans. The 

problem encountered prior to INChI is that the 

The goal of this research is to create a 

chemical naming system that would allow 

computers to uniquely identify a chemical, 

regardless of how it is drawn based entirely on the connectivity of the molecule – 

i.e., what atoms are connected to what 

other atoms. 

IUPAC 

The IUPAC-NIST Chemical Identifier 

(INChI) 

The need for a uniform and open standard that could be adopted by the 

entire chemical community prompted 

the NIST/IUPAC project to develop a 

chemical identifier. The aim of the 

project was not to create another 

naming system for normal communication, but rather to create a naming 

system that would allow computers to 

uniquely identify a chemical. 

CH3 H3C

H3C

CH3

CH3

CH3

trans cis unspecified
140

data retrieval was often dependent upon the way the molecule had been drawn. There is often a 

need to distinguish between the cis and trans form, and often a need to search for all possible 

forms, including cases where the configuration of the molecule was not known or it was known 

that a mixture was present. 

The approach taken in developing INChI is a layered approach. This allowed as much information 

as was known to be specified, the search could be performed only on the information known, and 

the search could be stopped with less than full information. Thus, in the case above, a search for 

the cis isomer could be allowed to stop when it matched the connectivity or continued to find only 

the molecules that matched the geometric isomer. The methodology of INChI also conforms with 

the XML standards and the output of the method can be done in XML or in simple text. 

The IUPAC NIST Chemical Identifier has been released for beta testing. The current version has 

been adopted by PubChem at NIH, is an integral part of the Chemical Markup Language (CML) 

standard, and has been integrated by ACD Labs in their widely used commercial drawing program, 

ChemSketch. In addition INChI will be integrated into the next version of the Chemistry WebBook 

so that anyone with access to the Internet can make use of this technology. 

Selected Data Activities in FY 2004 

(Described in more detail elsewhere in this report) 

Automotive and Aerospace 

 

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) 

Propulsion Systems Demand Accurate Property Data 

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

(838), I. M. Abdulagatov (Russian Academy of Sciences), and P.C. Andersen (2B Technologies) 

Combustion Simulation Databases for Real Transportation Fuels: A New Community Collaboration 

T. C. Allison, D. R. Burgess, Jr., J. W. Hudgens, W. Tsang, and J. A. Manion (838) 

Chemical and Allied Products 

IUPAC Partnership Develops Standards and a Data Retrieval System for Ionic Liquids 

J.W. Magee, J.A. Widegren, D.G. Archer, S.L. Outcalt, M.Frenkel, R.D. Chirico, Q. Dong, A. Laesecke, 

M.O. McLinden, R.A. Perkins (838), K.N. Marsh (U. of Canterbury, New Zealand), B.-C. Lee (Hannam U., Korea), and E.M. Saurer (U. of Notre Dame) 

Second Industrial Fluid Properties Simulation Challenge 

R.D. Mountain, D. Friend, R.D. Johnson, A.M. Chaka, F. Case (Case Scientific), D. Frurip, J. Moore, J. Olson 

(Dow), J. Golab, (BP Amoco), P. Gordon (ExxonMobil), P. Kolar (Mitsubishi Chemical), R.Ross (3M), and 

M. Schiller (DuPont) 

Systematic Validation and Improvement of Quantum Chemistry Methods for the Prediction of Physical and Chemical Properties 

C.A. Gonzalez, R. D. Johnson, and K.K. Irikura (838) 

Thermodynamic Reference Data - REFPROP 

E.W. Lemmon, M.O. McLinden, A.H. Harvey, and R.A. Perkins (838) 

ACD
141

Forensics and Homeland Security 

Explosives on Surfaces: A Sticky Problem T.J. Bruno (838) and K.E. Miller (U. of Denver) 

Raman Libraries 

S.J. Choquette (839) 

Health and Medical Technologies 

NIST Launches New HIV Bioinformatics Database 

T. N. Bhat(831); and A. Wlodawer (NIH-National Cancer Institute) 

Thermodynamics of the Redox Reaction for Fatty Acid Desaturase 

V. Reipa and V.L. Vilker (831); J. Shanklin (Brookhaven National Laboratory) 

Mass Spectroscopy in Health and Environmental Science 

S.E. Stein, P. Neta, and L. Kilpatrick (838) 

Industrial and Analytical Instruments and Services 

AnIML – Analytical Information Markup Language 

B. A. Schaefer (University of Kaiserlautern, Germany); D. Poetz (Fachhochschule Wiesbaden, Germany); 

A. D. Nguyen and G. W. Kramer (839) 

AMDIS – Automatic Mass spectral Deconvolution and Identification Software 

G. Mallard, S. Stein, and O. Toropov (838) 

Microelectronics 

Thermophysical Properties of Gases used in Semiconductor Processing 

J.J. Hurly, K.A. Gillis, and M.R. Moldover (836) 

Measurement Science for Future Standards and Technologies 

A New Monte Carlo Application for Complex Sample Geometries 

N. W. M. Ritchie (837) 
142
143

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5000 10000 15000

Relative number of cells

Cell Area (μm2)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5000 10000 15000

Relative number of cells

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5000 10000 15000

Relative number of cells

Cell Area (μm2)

Cell morphology on reference fibrillar 

collagen films. (Black) prepared 

2003; (Grey) prepared 2004. 

13. Measurement Science for Future Standards and Technologies 

... 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. 

Metrology Tools for Quantitative Cell Biology 

J.T. Elliott and K.J. Langenbach (831) 

Cell-based assays are utilized extensively in the biotechnology and 

pharmaceutical industry during multiple phases of product and drug 

development. Despite the widespread use of these assays, there has 

been little effort in developing metrics and standards to validate cell 

lines and the culture conditions before their use in an experimental setting. The absence of these validation procedures can cause complications when intra- or inter-laboratory data comparisons are required to 

fully interpret experimental results. The NIST research in quantitative 

cell biology has three dimensions: 1) development of indicator cells that express green fluorescent protein to report a 

cellular response, 2) development of highly reproducible extracellular matrix protein thin film substrates for use as reference cell culture substrates, and 3) automated fluorescence microscopy and image analysis methodologies for quantifying a cellular response. 

Indicator cells under development contain an artificial gene encoding Green Fluorescent Protein (GFP), causing it to 

generate its own fluorescent signal if a property in the culture/material induces a specific cell response. As proof of 

principle, a mammalian fibroblast cell line has been engineered to fluoresce during proliferation using an artificial GFPtenascin-C reporter gene. Rigorous biochemical studies designed to 

validate cellular biomarker response and to ensure the robust nature of 

the engineered cell line have confirmed that the activation of intracellular events associated with proliferation in these cells correlates with 

induction of cytoplasmic GFP. 

In order to reproducibly control the adhesion substrate for cells, NIST 

has developed a series of biomimetic materials based on highlyreproducible ultra-thin films of biologically relevant extracellular matrix (ECM) proteins adsorbed onto alkanethiol self-assembled 

monolayers. The physical properties of these thin films and the cellular response to these films have been extensively evaluated to validate 

their biomimetic nature and their reproducibility in generating a cell 

response. The figure shows the population distribution of cell morphology on the reference fibrillar collagen films prepared one year 

apart (black vs. grey). This precise level of validation is critical to 

stakeholders for establishing the utility of the collagen thin films as 

reference cell-growth substrates. 

CSTL leverages expertise in material and biological sciences toward 

meeting the metrology needs for 

the biotechnology and pharmaceutical industries. 
144

fibronectin

reduced fibril co llagen

fibrillar co llagen

co ntrol cells 0%

25%

50%

75%

100%

Relative number of cells

Fluorescence Intensity (A.U)

fibronectin

reduced fibril co llagen

fibrillar co llagen

co ntrol cells 0%

25%

50%

75%

100%

Relative number of cells

Fluorescence Intensity (A.U)

Histograms of GFP intensity from indicator cells on different reference ECM surfaces. The highest level of GFP expression occurred on fibronectin surfaces. Inset - phase 

and fluorescent pictures of GFP expressing cells. 

NIST has adopted automated fluorescence 

microscopy and image analysis as the primary tool for rapidly quantifying how indicator cells respond to changes in culture 

conditions. Cells are stained with a novel 

two-color staining method developed in our 

laboratory which allows measurement of cell 

density, cell morphology, and level of response (GFP signal intensity) in cells. The 

identification of this high-contrast and robust 

staining procedure for fixed cells will be 

useful to any research group working to address how culture conditions, extracellular 

matrix modification, and biomaterial substrates influence cellular behavior. Histograms of indicator cell GFP intensity on different reference ECM substrates (inset: 

phase contrast and fluorescent image of indicator cells on reference ECM surface) are 

shown in the figure. The highest level of 

GFP expression occurs when the cells are 

cultured on a fibronectin surface. 

Critical issues such as how cryopreservation and prolonged culturing influence cell behavior are being evaluated. In 

addition, future emphasis will involve the development of additional reference materials and protocols for standardizing 

measurements of cell-material interactions with the hope of bridging the gap between knowledge generation by basic 

scientists and product development in industry as well as addressing FDA concerns about quantitative cell measurements. 

Protein Quantitation 

N. Dodder and D.M. Bunk (839)

Proteins are measured in the clinical setting to assist in the 

diagnosis of a variety of diseases including heart attacks 

and cancer. Currently, the majority of these tests are performed using immunoassays. Although sensitive, fast, 

and relatively inexpensive, the specificity, accuracy, and 

precision of immunoassays can be a problem and necessitates a need for method validation through protein-based 

reference materials and methods. Two reference methods currently under development 

at NIST are based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) 

and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. 

Initial studies explored the quantitative capabilities of MALDI-TOF mass spectrometry 

for the small protein human insulin. Investigations were made in sampling issues, such 

as the creation of homogenous sample spots, and in the automated use of the mass spectrometer, programmed to search sample spots to produce uniform sampling. Based on 

this research a quantitative MALDI-TOF method was developed for human insulin, 

using pig insulin as an internal standard. The linearity, dynamic range, and error of this 

technique were acceptable. However, further studies using larger proteins revealed 

limitations of this approach, particularly the limitations in finding internal standards that 

would be applicable for the measurement of large proteins. 

CSTL researchers investigate new approaches to 

quantitative protein mass spectrometry to meet 

the need for new reference methods and materials 

to support clinical protein measurement. 

Structure of Insulin: 

source Protein Data Bank
145

An alternative approach is currently under investigation, using some of the methodologies developed in the growing 

field of proteomics. Instead of measuring intact proteins, the peptides produced from the proteolytic digests of intact 

proteins are measured. Using this proteomics-based approach, we are developing methods utilizing both MALDI-MS 

and LC/MS/MS for quantitative protein measurements. 

Because proteolytic digests, particularly trypsin digests, are at 

the core of our proteomics-based approach, we have devoted a 

large effort to understanding the practical nature of trypsin 

through fundamental studies of how experimental factors affect 

trypsin digests of analyte proteins. Guest Researchers from the 

United Kingdom (LGC) and Germany (PTB) have collaborated 

on this research effort. Successful quantitative measurements 

have been made on serum C-reactive protein using this approach. 

As the field of proteomics matures, more protein biomarkers will be discovered and used for clinical diagnoses. New 

immunoassays for protein biomarkers will require validation through more metrologically-sound methods, particularly 

methods based on mass spectrometry. Furthermore, accurate and precise protein quantification may help answer fundamental biological questions regarding protein expression and its relation to the genome and environment. Additionally, the techniques developed for protein quantification can also be used in the areas of drug discovery and biotechnology. 

We are developing a quantitative method for serum transferrin using a combination of ion-exchange chromatography, 

tryptic digestion, and MALDI-MS. A quantitative method for serum transferrin will be an important component in the 

on-going research at NIST to develop methods for serum iron 

speciation because transferrin is one of the principle serum iron 

transport proteins. We will also continue the development of a 

proteomics-based reference method for human C-reactive protein, a clinical biomarker for mild to severe inflammation as well 

as for cardiovascular disease. After further refinement of the Creactive protein reference method developed thus far, a validation study utilizing patient serum samples is planned. 

Nanobiotechnology: Nanoscale Chemical Reactions and Separations 

L.E. Locascio, W.N. Vreeland (839), A. Jahn, and M. Gaitan (EEEL) 

This project focuses on the development of nanoscale structures to facilitate the performance of ultra-small volume chemical reactions and 

separations. The work is associated with the Single Molecule Manipulation and Measurement NIST 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. 

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-sized structures 

for controlled chemical reactions using picoliters of reagents. 

Fabrication of microfluidic systems to facilitate the rapid and reproducible formation of liposomes with encapsulated 

fluorescent molecules: 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 made using bulk techniques range in size from 50 nm to tens of micrometers encapsulating volumes that are measured in attoliters to picoliters. Water-soluble molecules can be readily incorporated into the 

liposomes upon formation. The ultimate goal of our work is to use liposomes as discrete packages to sequester very 

C-Reactive Protein

Source: Protein Data Bank 

Ongoing research at NIST includes the 

development of: 

• a quantitative mass spectrometric 

method for serum transferrin, and 

• a reference method for C-reactive 

protein and its validation. 

NIST researchers develop nanometer-sized structures that can ultimately be incorporated into microsystems (microfluidics and microelectro-mechanical systems (MEMS) 

for use in studying the behavior of 

very small numbers of biological 

molecules with fine control. 
146

(a) White-light image of hydrodynamic focusing of IPA by 

buffer streams. Fluorescence images of (b) membranetracer fluorophore in IPA stream and (c) carboxyfluorscein 

(CF) in buffer streams. 

Silicon/glass microchannels have trapezoidal cross sections 

with the following dimensions; depth = 40 μm, maximum 

width = 200 μm, minimum width = 147 μm. 

small amounts of reagents in order to finely control their reaction. For this purpose, two characteristics of the liposome 

population are critical – the liposome size and the number of encapsulated molecules contained inside each liposome. 

Ideally, we would like all liposomes in a given population to be identical with a diameter of approximately 100 nm and 

containing one encapsulated molecule. However, liposomes prepared from bulk techniques generally exhibit a very 

large polydispersity with either uncontrolled or unpredictable encapsulation efficiency. For example, liposomes made 

in our laboratory using established techniques range in size from 70 nm to 200 nm in the same preparation. 

Demonstration of single-molecule encapsulation inside liposomes: Recently, we have demonstrated the automated and 

controlled formation of liposomes in microfluidic systems. In this work, we hydrodynamically focus a stream of lipid 

tincture at a microchannel cross-junction between two aqueous buffer streams. In a typical procedure, isopropyl alcohol 

(IPA) containing the dissolved lipids flows through the center inlet channel, and an aqueous phosphate-buffered saline 

solution flows through the two side inlet channels as shown in Figure 1a. When the two liquid phases come into contact, the IPA rapidly diffuses into the aqueous phase and vice versa. A fluorescence image of the liposome formation 

process is shown in Figure 1b where the IPA solution containing lipids also contains fluorescent dye that intercalates 

into the formed liposome membrane. Immediately downstream of the cross intersection (to the right in the image), the 

fluorescent intensity of the center stream increases indicating liposome formation (the quantum efficiency of the fluorescent dye in this experiment increases upon incorporation into a lipid membrane). The lipids self-assemble where the 

concentration of alcohol and buffer mixture is at a critical condition where lipids are no longer soluble. The flow rates 

of the alcohol and buffer channels are adjusted to control the 

degree of hydrodynamic focusing influencing both the dilution rate 

and the shear stress at the fluid/fluid interface. We have determined that changing this parameter allows us to maintain 

unprecedented control over the liposome size and homogeneity. 

After formation, the liposomes flow downstream for collection as a 

tightly focused stream owing to the low Reynold’s number laminar 

flow typical of microfluidics and the low diffusion coefficient of 

liposomes. 

This work 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. This year, we 

have made considerable progress toward packaging single molecules, 

and performing controlled reactions with a few molecules. Future 

work will involve further characterization of encapsulated single 

DNA and RNA molecules so that we can study their behavior one at 

a time. 

The NIST research team discovered that 

the characteristics of fluidic flow in a 

micrometer-scale channel can be used to 

precisely control the distribution of 

chemical conditions and mechanical 

forces so that they are constant on a 

length scale equivalent to that of a liposome. Hence, forming liposomes in micrometer-scale flow field results in more 

homogenous conditions during liposome 

self-assembly and resultant liposome 

populations that are more uniform in 

size, hence of low polydispersity. 
147

(a

1

2

3

4

5 5 μm 1μm

(b (c

a) Steps 1though 4 show the 

preparation of Au lines as 

catalyst. Step 5 shows an Au 

line after growth of NWs. b) 

and c) Sapphire surface after 

growth of NWs. Large-scale 

assembly of NWs is seen in 

low (b) and high magnification images (c). 

Horizontal Growth and In Situ Assembly of Oriented Zinc Oxide Nanowires 

B. Nikoobakht and S.J. Stranick (839) 

Research in the area of nanoscopic materials 

has the potential for providing new classes of 

materials with performance and functionality 

not achievable by systems on large length 

scales. In the field of photonics, work is underway in the development of nanoscale light 

sources and detectors that are capable of operation at the single photon level. When coupled with the ability to manipulate and assemble these elements into hierarchical structures, nanodevices capable of 

performing sophisticated functions such as quantitative chemical measurement will be realized. Our recent efforts in 

this area have focused on developing methods for directed growth and assembly of semiconducting nanowires (NW). 

In the majority of semiconductor NW syntheses schemes, a thin catalyst film is used as the nucleation site/media for 

growth of NWs, resulting in standing NWs with diameters ranging from 20 nm to 120 nm. However, this approach has 

limited control over the diameter of the NWs making diameters smaller than 10 nm difficult to prepare. Perhaps more 

important for most device applications, NWs need to be assembled and aligned in the plane of the surface. To this end, 

we have developed a new technique for the growth of semiconductor NWs, which overcomes the limitations stated 

above. Using our approach, single-crystal NWs with adjustable diameters between 1 nm to 20 nm are grown horizontally (in the plane of the surface) along 

crystallographic directions on an a-plane 

sapphire surface. 

Our studies show that the crucial requirements for horizontal growth of NWs are the size of and the spacing between Au 

catalyst nanodroplets. To fulfill these geometric requirements, we have used thin Au lines (width < 200 nm) and/or 

lines of Au nanoparticles as nucleation sites for NWs growth (Figure 1a). Using a vapor-phase transport process at high 

temperature, each nanoparticle in the Au lines nucleates ZnO formation and becomes an NW. The figure is an example 

of the resulting oriented NW arrays formed using this procedure. 

This research has focused on the growth and 

manipulation of semiconductor ZnO 

nanowires. These methodologies are now 

being implemented in the fabrication of heterostructured (ZnO/GaN) NWs for use as 

nano-emitting sources (laser diodes) and 

nano-detectors (photodiodes) with emphasis 

on optical properties and performance suited 

for applications in sensing and spectrochemical analysis 

CSTL develops an approach that promotes the horizontal 

growth, in situ alignment, and predictable positioning of 

ZnO nanowires (NWs), allowing for the large-scale production and assembly of ZnO NWs and NW arrays. 

The methodology developed at NIST will provide an inexpensive and straightforward approach to the synthesis of 

semiconducting nanodevices (emitters and detectors) with 

multiple functionalities that can be integrated to form stateof-the-art measurement systems 

B. Nikoobakht, C.A. Michaels, S.J. Stranick, M.D. Vaudin, 

“Horizontal Growth and in situ Assembly of Oriented Zinc 

Oxide Nanowires”, Appl. Phys. Lett. 85, 3244 (2004). 
148

Nanowires for Chemical Sensing 

R. E. Cavicchi, C. B. Montgomery (836), P. M. Parthangal, and M. R. Zachariah (University of Maryland) 

Nanowires (NWs), defined as electrically conducting structures with crosssectional dimensions on the nanoscale, possess unique characteristics that 

make possible a host of new chemical sensing devices. Nanowires have a high 

fraction of their constituent atoms on the surface and therefore have electrical 

properties that are exquisitely sensitive to the chemical environment. The high 

aspect ratio of nanowires is also useful for producing high electric fields at the 

tips providing enhanced field-emission which may be utilized for speciesdependent ionization of gases. While carbon nanotubes (CNTs) are an example that has received much attention, it is now possible to make nanowires of 

other materials ranging from semiconductors such as Si, CdTe, or ZnO to a 

variety of metals. To realize chemical sensors, methods of synthesis of 

nanowires must be developed that are compatible with integration onto chips. 

Sensing approaches that take full advantage of the unique properties of these 

materials must be developed. 

We have pursued two methods for fabricating nanowires on sensor chip platforms. The first method takes advantage of recently developed methods for 

growing CNTs and NWs that start with a nanoscale metallic particle. For 

CNTs a chemical vapor growth is performed using a mixture of hydrogen and 

a carbon-containing species, such as methane, acetylene, or ethanol, with the 

substrate held at temperatures from 600 °C to 1000 °C. Nucleation occurs on 

the metal particle. For NWs the process is similar, but with different precursors. Novel to our approach is a vapor-phase synthesis that produces charged 

nanoparticles in an argon stream. Electric fields are used to size-select the 

nanoparticles and guide their deposition to specific locations on a substrate. 

Figure 1 a) shows an example of the use of an applied electric field on one of 

the pair of interdigitated comb electrodes to localize the deposition of Ni particles to one electrode, and 1b) shows CNTs localized to one electrode, produced by chemical vapor deposition (CVD) of acetylene using the Ni particles 

as nucleation sites. 

A second growth technique for nanowires takes advantage of our recent 

discovery that tungsten, when heated in the presence of a reducing gas, 

undergoes a significant change in morphology. We have developed a 

process for producing NWs of tungsten by a simple hydrogen plasma 

treatment of thin film or bulk tungsten that uses a much lower temperature (450 °C) than is required for the vapor deposition methods described 

above. Thin films of tungsten treated in this way are converted to NW 

"grass" as shown in the figure to the left. We have found that treatment 

of smooth tungsten filament wire results in a bristle coating of NWs, 

which may also be useful for applications where field emission is used. 

Tests have shown these NW films to be sensitive to volatile organics. 

We are currently exploring treatments of the nanowires, including templated growth of other materials on CNTs and 

NWs. We are exploring the limits of field-guided deposition of nanoparticles, including a novel self-assembly approach 

that uses the concentration of electric fields at the tips of NWs. These new materials will be integrated with existing 

silicon micromachined sensing platforms in our group to perform chemical sensing via electrical measurements at a 

wide range of temperatures. 

(a) 

(b)

The research team seeks to 

create nanowires and nanomaterials for a new generation of chemical sensors with 

enhanced sensitivity and 

chemical selectivity 
149

High Performance Oxide and Polymer Nanostructures for Advanced Solid State Chemical 

Microsensors 

K.D. Benkstein, C J. Martinez, G. Li, D.C. Meier, S. Semancik (836), T. Mallouk (Penn State), and V. Dravid 

(Northwestern) 

Efforts underway at NIST, and through collaborations with 

two universities, are aimed at developing nanoparticles, nanofibers, nanowires, nanotubes, and related structures of oxides 

and polymers for use in nanosensor arrays and microanalytical 

systems. These nanomaterials are being studied as signal 

transducers for chemical sensors and as elements for filtering 

and preconcentration of analytes prior to detection. The approach is to assemble hierarchical structures for chemical 

sensing applications from single nanostructures and various 

nanocomposite materials. These well-defined nanostructures 

will increase the surface area and active interfacial sites for 

adsorption and reaction of gas phase analytes, thereby leading 

to more sensitive chemical measurements. These structures are 

also designed to enhance diffusion of the target molecules to 

functional sites by introduction of various scales of micro- and 

nano-porosity. 

Specific materials forms being studied include SnO2 microshells, TiO2 nanoparticles, porous SiO2, nanofiber and 

nanowire polyaniline, nanowire oxides, and pure and doped 

SnO2 sol gels dispensed by dip pen nanolithography. The 

SnO2 microshells are formed via a solution phase, layer-bylayer deposition of SnO2 nanoparticles on sacrificial polystyrene spheres. These solutions are deposited on microheater 

platforms and the polystyrene spheres are removed by rapid 

heating to leave hollow, nanoporous SnO2 shells with ultrathin 

walls. The microheaters also provide electrical contacts to the 

SnO2 microshells for chemical sensing measurements. The 

SnO2 microshells have exhibited sensitivity factors as high as 

50 for detecting 1000 μmol/mol methanol in air. Porous silica is being developed as a high-area support material for 

microscale preconcentrators and filters. This mesoporous material is formed by heating silsesquioxane/block copolymer 

blends to produce a highly porous SiO2 structure. While much of our sensing research has focused on semiconducting 

oxides, conducting polymers are also now being investigated to expand the range of analytes that can be sensed by multielement microarrays. We have found that polyaniline nanofibers respond more than seven times faster than denser 

polyaniline films, and through a collaboration with Penn State, we are exploring the sensitivity and speed enhancements 

possible with polyaniline nanowires. Nanowires and nanotubes of 

polyaniline, SnO2, and TiO2 are all being studied as fundamental 

building blocks for constructing nanoarrays capable of analyzing 

complex chemical mixtures. Related oxide materials are being investigated with Northwestern, where dip pen nanolithography has been 

employed to pattern SnO2 nanomaterials. 

 Future studies at NIST and at collaborating institutions will develop 

more reproducible processing methods and explore the fundamental 

mechanisms responsible for sensing enhancements realized through 

nanoengineering. Methods of manipulating nanostructures, particularly single particles, wires, and tubes, onto microdevice platforms to 

enable electrical, and hence sensing, measurements, remains a challenge and will also be a focus of our studies. 

The CSTL-led research team is developing 

nanoengineered materials for microsensors 

and microanalytical systems to increase sensitivity, selectivity, and stability of chemical 

detection and monitoring. 

TEM image of SnO2 microshells formed on 

sacrificial polystyrene spheres with layerby-layer assembly of nanoparticles 

Higher sensitivity, stability, and reproducible fabrication of sensing materials 

are critical to next-generation sensing 

devices. The improved sensitivity, selectivity, and response time that can be 

attained by proper assembly of nanobuilding blocks is expected to impact 

many application areas including alarm 

triggers for counter-terrorism, trace gas 

detection in space exploration, and the 

monitoring of gaseous bioproducts for 

medical diagnostics. 
150

Controlling "Injection Barriers" into Prototype Molecular Wires Through Substrate Coupling 

Chemistry 

S. W. Robey (837), R. D. Van Zee, and C.D Zangmeister (836)

The drive to introduce organic molecular materials into electronic device applications (organic or molecular electronics) 

is motivated by a number of potentially attractive features, such as ease of fabrication, ability to fabricate on flexible 

substrates, and the wide extent to which organic materials can be functionalized via organic synthetic methods. A range 

of applications is foreseen for organic field effect transistors and light emitting diodes including, for instance, flexible 

displays and other low-cost flexible electronics. 

In addition to replacing inorganic semiconductors in more or less conventional device architectures, organic systems are 

also of interest in the more speculative, and potentially more revolutionary, area of “molecular electronics”. Here, it is 

envisioned that the nonlinear characteristics of individual molecules, or small ensembles, will provide the required device functionality, allowing low-cost chemical synthetic methods to replace, at least partially, multi-billion dollar semiconductor fabrication lines in the production of nanoscale device structures. 

In either application format, charge injection at a molecule-contact interface plays a vital role in controlling transport 

and, thus, potential device performance. Interfacial charge injection is dictated by chemical bonding, and the resulting 

band line-up between the Fermi level of the contact and transport levels of the molecule. Information relating to band 

line-up is difficult to obtain using conventional techniques in the case of the single-molecule length-scale systems of 

interest in molecular electronics. We have employed a combination of one-photon (He I resonance excitation) and twophoton photoelectron spectroscopy, using sub-picosecond Ti:sapphire laser-based excitation, to determine the electronic 

structure, including injection barriers to both occupied and unoccupied levels, of oligio(p-phenylene-ethynylene)thiol 

(OPE) self-assembled monolayers, a system that has become a benchmark for theoretical and experimental studies in 

the area of molecular electronics. 

The majority of studies of covalently bound monolayers on 

metallic surfaces involve thiol-coupling (R-SH) chemistry. 

Self-assembly of monolayers utilizing thiol chemistry is 

known to form robust, reproducible monolayers on a variety 

of metallic surfaces. However, it is important to characterize 

how the linker group affects the electronic spectra and consequently the band line-up of adsorbed monolayers. Thus, 

we have undertaken a study of understanding how the band 

line-up varies as a function of changing the linking chemistry between the molecule and the surface. One-photon photoelectron spectra in the figure shows that the substitution of 

the thiol-linker for isocyanide (R-NC) in OPEs adsorbed on 

Au shifts the position of the occupied and unoccupied states 

relative to the Fermi level by approximately 0.5 eV. Such a 

large variation will have a major impact on charge injection 

in molecular systems. Models for important aspects of the 

bonding in these two systems to Au have been developed 

that help explain the observed behavior. 

This research team will build upon previous studies of understanding the effect molecular structure and linking-group 

chemistry have on controlling band line-up in covalently 

bound molecular systems. Our future work will focus on 

varying the metal onto which the molecule is adsorbed, as 

well as alkali metal doping of monolayer films. 

One-photon photoelectron spectra of OPEs adsorbed on Au showing shift in band line-up by 

varying the linking group from thiol (black line) to 

isocyanide (red line). 

10 8 6 4 2 0

0.0

0.2

0.4

0.6

0.8

1.0

 OPE-NC

 OPE-SH

Intensity (a.u.)

Binding Energy (eV)

The results of the CSTL research add important insights to the factors controlling band 

alignment at metal-organic interfaces, a parameter that plays a critical role in potential 

applications of organic materials in emerging 

electronic technologies. 
151

Quantitative Analysis of Submicrometer Particles in the Scanning Electron Microscope (SEM) Utilizing the ζ Factor Approach 

J.A. Small, N.W.M. Ritchie, and J.M. Davis (837) 

The analytical electron microscopy (AEM) approach for the quantitative analysis of ultrafine particles, 0.1 nm to 500 

nm in size, involves the application of an elemental ratio procedure known as Cliff-Lorimer (CL). This procedure was 

designed for the AEM analysis of “infinitely” thin samples, for which there is negligible x-ray absorption. The basis for 

the CL approach is given in the equation to the right: Where kab is a proportionality constant 

calculated, using materials of known composition, for a given instrument at a given voltage, 

Ca and Cb are the concentrations of elements a and b, and Ia and Ib are the measured x-ray 

intensities. 

This investigation was undertaken to determine if the CL approach is applicable to the analysis of ultrafine particles in 

the SEM at 25 kV to 30 kV for which x-ray absorption is not negligible. In this study, a modified CL analysis procedure 

was employed that includes a correction for x-ray absorption known as the ζ factor; this factor was defined in Ultramicroscopy in 1996. 

During the past year we have made a series 

of measurements on glass shards of known 

composition that had been ground to produce a large number of ultrafine particles. 

The particles were analyzed at 25 keV. 

The results are shown in the figure, which 

plots the Al concentration analyzed by both 

the CL method and the modified CL that 

includes the ζ factor. The Al concentration 

from the CL method at a mass fraction of 

0.063% has a negative bias compared to the nominal value of 0.074% as a result of not correcting for the absorption of 

the Al x-rays in the particles. In comparison, after the incorporation of the ζ factor into the CL method the mass fraction 

of Al percent is 0.075%, effectively removing the observed bias and bringing the average concentration more in line 

with the nominal value. 

This study represents one of two methods we are currently 

investigating to improving the quantitative analysis of the ultrafine particles. The other approach involves reducing the 

accelerating voltage of the scanning electron microscope 

(SEM) to 5 kV or less for analysis. The rational for this approach is that the x-ray generation and emission volumes at 

the low voltage will be contained within the particle volume to 

a much greater degree than excitation at a higher but more 

conventional voltage such as 15 kV. Future plans involve the 

comparison of these two methods and their effectiveness for 

the quantitative analysis of ultrafine particles. 

Ca/Cb = kab * Ia/Ib

Plot of the Al concentration in 

ultrafine glass particles analyzed by both the CL method 

and the modified CL, which 

includes the ζ factor. 

Traditionally ultrafine particles have been 

analyzed in analytical electron microscopes, 

which often require large capital investments 

in excess of $1M and have difficult sample 

preparation procedures. The SEM method 

described here could potentially offer a more 

economical approach. 

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

100 200 300 400 500 600 700

size nm 

Al CL

Al ζ

Average 

Al CL = 0.0626%

Al ζ = 0.0748%

Nominal Mass Fraction = 0.0741 % 
152

A New Monte Carlo Application for Complex Sample Geometries 

N. W. M. Ritchie (837) 

Performing quantitative microanalysis on micrometer-sized 

particles has always been a challenge. The volume of the 

particle is typically smaller than the electron beam excitation 

volume and x-ray absorption corrections are complicated by 

surface topology. Some researchers have approached this 

problem by approximating the particle as a simple geometric 

shape such as a cylinder, a rectangular, or triangular prism. 

While this approach is better than applying bulk correction methods, it may be possible to combine topological measurements from multiple imaging detectors to build a more accurate three-dimensional model of the unknown particle. 

This model could then become the input sample structure for a Monte Carlo simulation. The Monte Carlo simulation 

could be compared to Monte Carlo simulations of bulk references and the result could be iterated in a manner similar to 

the iterative correction processed used by the standard ZAF correction* scheme. The result is likely to be more accurate 

quantitative results. However, this scheme relies on the availability of a Monte Carlo model that can handle complex 

sample geometries. Our work seeks to develop a library of Monte Carlo simulation routines capable of handling samples of arbitrary geometric complexity. 

We developed and tested a Monte Carlo simulation implemented in platform-independent 

Java code. We have evaluated various algorithms for electron elastic (see figure) and inelastic scattering cross section, electron energy 

loss, fluorescence yield and mass absorption 

coefficient. For each algorithm class, we selected the one that we determined produced the 

most realistic results. These algorithms have 

been implemented into a model in which the 

sample is represented by instances of a generic 

Shape interface. (Shape refers to a Java interface – a contract between a class’s user and 

implementer). The Shape interface represents 

samples of arbitrary complexity with sufficient 

detail for the purposes of this model. Implementations of the Shape interface have been 

created for basic shapes such as spheres, 

blocks, and the volume defined by the intersection of an arbitrary number of directed 

planes. In addition, implementations of the 

Shape interface have been created to represent 

the union of two or more Shapes and the difference of two Shapes (the volume of Shape A 

minus the intersection between Shape A and 

Shape B). By combining these Shapes programmatically, samples of arbitrary complexity can be built from primitive Shapes. 

Assigning particles to a descriptive class through quantitative microanalysis is hampered by morphologically induced 

particle-to-particle variance. We anticipate that by better modeling the shape of the particle we will be able to reduce 

particle-to-particle variance and thereby improve our ability to differentiate particles of similar but different materials. 

In the coming year, we plan to use the results from this Monte Carlo simulation to develop and evaluate more computationally efficient analytical expressions for quantifying particulate samples. 

*ZAF Correction: Conversion of the X-ray intensities to concentration based on measurement of the pure element intensities and composition of corrected factors for “atomic number effect” (Z), the “absorption correction” (A), and the 

“fluorescence correction” (F). 

20 keV

0%

10%

20%

30%

40%

50%

60%

70%

0 20 40 60 80 100

Atomic number

Backscatter Yield

Rutherford

NIST-Mott

Czyzewski

Experiment

The modeled backscatter yield for various different implementations of the electron elastic scattering cross-section compared with published measured values from Heinrich. 

Rutherford corresponds to a simple screened Rutherford 

cross-section; NIST-Mott and Czyzeweski are different implementations of the Mott cross-section. The Czyzewski crosssection reproduces the experimental results most accurately 

across the full range of atomic numbers. 

The CSTL research seeks to develop a library 

of Monte Carlo simulation routines capable of 

handling samples of arbitrary geometric complexity for performing quantitative microanalysis on micron-sized particles. 
153

Improved Energy Stability in the NIST Microcalorimeter X-Ray Detector 

T. Jach, J.A. Small, and D.E. Newbury (837) 

Microcalorimeter x-ray detectors make up a 

new technology that combines some of the 

positive features of wavelength dispersive 

(high resolution) and energy dispersive (parallel detection over a wide energy range) detectors that have gained broad acceptance in the 

microanalysis community. Microcalorimeters 

which use a transition edge sensor (TES) have 

proven to be effective over energy ranges of 10 

keV or more in applications such as x-ray fluorescence analysis with electron microscopes.

TES microcalorimeter x-ray detectors have 

successfully demonstrated energy resolutions 

better than 5 eV. However, serious drifts in 

energy scale over extended counting times 

have set limits on both the long-term resolution and the calibration of these detectors. This 

is because the operating point of the detectors 

is the middle of the superconducting-normal 

phase transition. The successful operation of a 

microcalorimeter as an x-ray detector puts 

considerable constraints on the stability of all 

the electrical and thermal inputs to the instrument. Previously observed drifts of >10 eV/h 

have been reduced to 1 eV/h to 2 eV/h. This 

improved stability, shown in the figure, has 

resulted in the observation of x-ray fluorescence linewidths of 12 eV to 15 eV over a 6 h 

time period. 

The detector is cooled to a substrate temperature of only 70 mK and maintained at its operating point by a complex 

feedback control system connected to a large superconducting magnet. We carried out a careful analysis of the performance of all the elements in the control system including the response function of the magnet under typical conditions of operation. We determined that the desired stability and performance of the detector required control of its substrate temperature to a precision of 23 μK, that is, a variation of less than 5 parts in 10,000. By careful modification of 

the control circuitry, we have been able to realize this degree of temperature stability of the substrate. The energy scale 

of the spectra is now observed to be stable to within about ≤1 eV/h over a period of hours, under operating conditions in 

which a linewidth of 12 eV to 15 eV is readily obtained. X-ray spectra acquired over long durations under these conditions of substrate temperature stabilization show vastly improved stability and resolution. 

Additional measures to stabilize the operating temperature 

are possible. Once realized, we can carry out a demonstration of the microcalorimeter detector with an electron microscope for quantitative microanalysis. We also anticipate replacing the actual detector element with higher 

resolution (< 4 eV) versions, which have been developed 

at NIST, Boulder. Combined with the achieved stability of 

the energy scale, we can start to look at characterizing 

chemical states of some elements by the energy of their 

fluorescence lines. 

The CSTL research team investigated the 

sources of energy scale drift in the NISTdeveloped microcalorimeter x-ray detectors 

and has addressed the most critical elements. 

Energy (eV)

850 900 950 1000 1050 1100 1150

Counts

0

1000

2000

3000

4000

Brass 10,000 s

Zn 2000 s

Cu 2000 s

 Cu Lα

Zn Lα

Cu Lβ

Zn Lβ

Cu, Zn, SRM Brass Spectra 

(total time 6 h) 

The drift in energy scale of the microcalorimeter 

x-ray detector has been a major limitation to the 

commercial development and marketing of this 

type of detector. There are currently a number 

of potential applications of this detector, particularly in the semiconductor industry. 
154

Rapid Searching of Spectrum Image Databases for Rare Events, or Finding the Needle in the Haystack 

When You Don’t Even Know You’re Looking for a Needle! 

D.S. Bright and D.E. Newbury (837) 

Characterization of the microstructure of materials often requires 

detection of rare features, such as naturally occurring minority/trace phases or unintentional particulate contamination. Xray mapping in the scanning electron microscope (SEM), a traditional tool for measuring elemental distributions with micrometer to nanometer spatial resolution, has recently been greatly 

enhanced by the development of x-ray spectrum imaging, in 

which a complete x-ray spectrum is stored at each pixel location 

visited by the beam. The silicon drift detector (SDD), described 

here in FY03 and whose development was aided by NIST Small 

Business Innovation Research (SBIR) grants, combined with digital signal processing enables x-ray count rates above 

100 kHz, permitting recording of useful x-ray spectrum images in 200 s or less. The resulting stream of 200 Mbyte 

image databases is creating a demand for software tools that are quick and efficient at locating features of interest. 

We have developed a software tool within the NIST LISPIX image processing platform that determines the 

MAXIMUM PIXEL SPECTRUM by finding the maximum value within each energy channel x-y plane and plotting 

this value versus energy. 

This new function is compared with the SUM 

SPECTRUM, similarly calculated by adding all 

values within a plane, as shown in Figure 1(a) for a 

spectrum image of Raney nickel, a methanation 

catalyst. Peaks in the SUM SPECTRUM correspond to common features in the x-ray spectrum 

image, as illustrated in Figure 1(b) where the aluminum-rich phases are highlighted. While these 

same peaks are found in the MAXIMUM PIXEL SPECTRUM, additional peaks can be recognized that correspond to 

rare events, down to the single pixel level, shown in Figure 1(c) for a chromium contaminant that appears at a single 

pixel, or 1/51200 for a 256x200 scan. Note that the rare chromium feature has been found despite being completely 

unknown to the analyst. 

The MAXIMUM PIXEL SPECTRUM has had an immediate impact in the microanalysis field. First publicly presented at the 

SCANNING 04 conference held in Washington in April 2004, the 

method was immediately adopted by a US manufacturer of microanalysis software systems, appearing in this vendor’s system at the Microscopy and Microanalysis Conference in August and in their advertisement in Microscopy Today in September 2004. Other vendors are rapidly incorporating the 

MAXIMUM PIXEL SPECTRUM as a feature in their spectrum imaging software. We anticipate that the combination 

of SEM SDD x-ray spectrum imaging and derived spectrum image processing tools will have a broad impact in materials analysis, supporting technology, physical and biological science, and forensic applications. The MAXIMUM PIXEL 

SPECTRUM and the SUM SPECTRUM are members of a class of transformations known as “derived spectra” to distinguish them from the true spectra recorded in the spectrum image. We plan to investigate additional algorithms for 

derived spectra to seek software tools that can aid the analyst in other aspects of x-ray spectrum imaging. Other microanalysis spectroscopies, such as electron energy loss and Auger electron, may also benefit from derived spectrum tools. 

CSTL researchers have developed the 

Maximum Pixel Spectrum, a software tool 

that enables rapid searching of SEM x-ray 

spectrum image databases to detect rare features, even if the analyst does not know in 

advance which elements are present. 

This software tool is available free at 

http://www.nist.gov/lispix/ 

Channel

Intensity

NiKα

NiKβ

CrKα

NiL AlK

CK

SiK

ClKα

KKα CuKα OK

FeKα CuL

Log SUM

MAX

Al Cr 50 μm

(b) (c)

(a) (a) SUM SPECTRUM (upper trace, log 

intensity) and MAXIMUM PIXEL 

SPECTRUM (lower trace, linear intensity). 

(b) A major feature (aluminum-containing 

areas recognized from peaks in the SUM. 

(c) A rare feature, chromium, detected at a 

single pixel (inside circle) from the 

MAXIMUM PIXEL SPECTRUM. 
155

Evanescent Wave Cavity Ring-Down Spectroscopy (EW-CRDS) of Surface Hydroxyl Groups on SiO2

A.C.R. Pipino (836) and I.M.P. Aarts (Eindhoven U. of Tech., The Netherlands) 

The SiO2 surface is ubiquitous in science, technology, and the environment. Whether used as a catalytic or chromatographic support or as the 

substrate for atomic layer deposition, the SiO2 surface displays reaction 

chemistry that is largely controlled by the presence of surface silanol 

(SiOH) groups. Yet ambiguous results are often obtained in spectroscopic 

studies of silica gels or powders, which increase surface area to enable 

detection of the weak surface SiOH signals. To realize a decisive 

diagnostic probe of the planar SiO nostic probe of the planar SiO2 surface, we use evanescent wave cavity ri 2 surface, we use evanescent wave ng-down spectroscopy (EW-CRDS) that is a 

variant of the gas-phase CRDS technique employing a miniature, ultra-low-bulk-OH fused-silica optical resonator with 

total-internal-reflection (TIR) mirrors. 

Using EW-CRDS we have obtained vibrational combination band spectra of 

surface SiOH and adsorbed water on ultra-smooth amorphous silica in the 

8000 cm−1 region. By obtaining spectra at both s and p polarizations, we are 

able to identify the dipole orientation of the SiOH group for each observed 

spectral peak. Remarkably, we find evidence for crystalline character for both 

SiOH and adsorbed water layers, where the latter suggests the formation of an 

ice-like structure at room temperature. The combination band spectra for an 

OH mode of surface SiOH under dry nitrogen gas is shown for s and p polarizations are shown in the figure to the right. Note that the maximum optical 

loss for these weak surface spectra is only 2×10-6, which illustrates the very 

high sensitivity of EW-CRDS. Moreover, two SiOH bands are observed, 

which are both highly polarized. The p-polarized (8118 cm-1) band indicates a 

mean orientation for the transition dipole that is perpendicular to the surface 

plane, while the s-polarized (8152 cm-1) band indicates a parallel transition 

moment. Such spectra are consistent with the existence of a crystalline surface 

phase, similar to a facet of crystalline silicon dioxide. 

One of four adsorbed-water peaks 

is shown in the figure to the left. 

Under dry nitrogen (no previous heating), adsorbed water is still present due 

to rather strong hydrogen bonding with the surface SiOH groups. Exposed to 

10% relative humidity, the water band increases strongly and sharpens 

slightly. However, when exposed to 50% relative humidity, the water band 

shows only a slight additional increase by contrast, suggesting the band intensity saturates at one-monolayer coverage. Furthermore, this band and the 

three other observed adsorbed-water bands are also highly polarized, indicating an ordered water layer supported by the underlying ordered SiOH layer. 

The existence of an ice-like layer on silica at room temperature has also been 

inferred from sum-frequency generation (SFG) measurements and predicted 

in recent theoretical studies. EW-CRDS provides detailed surface structural 

information with the potential for absolute surface coverage and absolute 

surface reaction rate determinations. 

This work is part of an on-going collaboration between NIST and the (TU/e) 

in the Netherlands. Originally developed at NIST, EW-CRDS is currently 

being used at TU/e to probe thin-film structure and growth processes, including growth of amorphous, hydrogen-doped 

silicon and atomic-layer-deposition processes. Other current 

applications of EW-CRDS involve studies of the liquid/silica 

interface and novel size-selected nanoparticle catalysts. 

The NIST-led research team demonstrates the capability of EWCRDS detection as a probe and 

diagnostic tool for investigating 

chemistries at interfaces. 

Shifts in water absorption

on SiO2 surfaces. 

S and P OH mode polarized 

absorption spectra for SiOH.
156

Precision Densimetry for Primary Temperature Metrology 

M.O. McLinden (838) 

Details provided in the Exploratory Research Section of this Report

Chiral Temperature Gradient Focusing 

D. J. Ross (839), K. Balss (836), K. Phinney, and W.N. Vreeland (839) 

Details provided in the Exploratory Research Section of this Report

Computational Chemistry Illuminates Atomistic Processes at Complex Interfaces 

A. M. Chaka (838) 

Details provided in the Chemical and Allied Products Section of this Report

Ab Initio Mass Spectrometry 

Karl K. Irikura (838) 

Details provided in the Exploratory Research Section of this Report

Invention of a New Class of Ultra-Fast, Ultra-Sensitive Mass Spectrometers for Kinetics, Reference 

Mass Spectrometry, and Homeland Defense Applications 

J. W. Hudgens (838) 

Details provided in the Exploratory Research Section of this Report 

Development of ICP-OES Determination of Phosphorus as a Primary Measurement Tool for 

Quantitation of Plant Deoxyribonucleic Acid (DNA) 

M.J. Holden (831), M. Winchester (839), J.R. Blasic, Jr., M. Dizdar, P. Jaruga, and Y. Tewari (831) 

Details provided in the Exploratory Research Section of this Report 
157

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 six years 80 exploratory research projects 

have been funded, and CSTL has invested close to $6M. 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. Exploratory research most often supports anticipated metrological needs or Future Measurements and Standards. This 

research is not yet applied to a specific industrial sector, it is infrastructural, and cross-cuts our customer-based programs. The work described in the following pages is a result of the research from FY 2004 funded projects. 

Optical Characterization of Organic Electronic Thin Films 

L.J. Richter (837), M.C. Gurau (837), D.M. DeLongchamp (MSEL) 

CSTL Program: Measurement Science for Future Standards and Technology

Organic electronic (OE) devices are projected to revolutionize 

integrated circuits through new applications that take advantage of 

low-cost, high-volume manufacturing, nontraditional substrates, and 

designed functionality. OE devices, which rely on custom designed 

organic molecules and polymers instead of crystalline inorganic 

material such as silicon, present the electronics industry with 

fundamentally different measurement challenges. For example, the 

structural order in ultrathin OE films, particularly at the buried 

film/gate-dielectric interface, is thought to be critical to the 

achievement of high performance. However, x-ray diffraction techniques, the ‘gold standard’ for structural determination, are difficult with very thin films. Additionally, as many target applications require flexibility in the OE film, true 

crystalline order (which gives rise to diffraction) may be undesirable. In this exploratory research project, optical techniques originally developed for the characterization of molecules adsorbed at surfaces are explored as tools for the 

structural characterization of thin films. 

Poly3hexylthiophene (P3HT) has emerged as a prototypical OE material. Transistors made from this material can 

achieve performance levels close to those of amorphous silicon. Data from traditional structural characterization tools, 

however, have not shown strong correlations with the electrical performance of P3HT films. Surprisingly, low relative molecular mass, highly 

crystalline films have poorer performance than high relative molecular 

mass films that appear highly amorphous. 

Shown in the figure to the right (a) are absorption spectra for a high relative molecular mass P3HT film before and after annealing. The fine 

structure that appears near 2.0 eV after annealing has been attributed to 

an electronic excitation that spans multiple chains and is indicative of 

good interchain order. Shown in the main panel of the figure (b) are infrared spectra probing the alkyl side groups (features at 2800 cm-1 to 

3000 cm-1) and the thiophene ring (823 cm-1). 

Infrared spectroscopy allows selective study of different parts of the polymer chain, but averages the information over the entire film. The alkyl side 

chains show little change upon annealing, indicating that, contrary to the 

behavior in bulk crystals, the alkyl groups do not drive the ordering of the 

thin films. Detailed analysis establishes that the alkyl groups are highly 

disordered. There is significant change in the thiophene ring feature, indicating that the chains adopt a more card-edge stacking structure upon annealing. The inset to Figure 1 (b) shows nonlinear vibrational spectra of the 

alkyl groups (SFG = sum-frequency generation). Unlike infrared spectros- Absorbance (arb)

1.5 2.0 2.5 3.0 3.5 4.0

unannealed

annealed

a

Photon Energy (eV) 

CSTL researchers demonstrate that 

vibrational spectroscopies can provide 

new insight into the structure of OE 

films and potentially may provide the 

basis for evaluation and monitoring of 

OE films by manufacturers. FTIR Absorbance (arb)

2800 2900 3000 3100 SFG Signal800 900

b

Wavenumber (cm-1) 
158

copy, the nonlinear spectroscopy is only sensitive to the surface of the thin film. The presence of the feature near 2960 

cm-1 indicates that, while the alkyl groups are disordered in the interior of the film, they are highly ordered at the surface. The combination of the three spectroscopies (visible, infrared, and nonlinear) provides a detailed picture of the 

evolution of the structure that can be related to the electronic properties of the film. 

The next challenges include correlating the spectroscopic results with direct 

measurements of the electrical performance of the films to establish the 

structural characteristics of high performance films. The optical techniques 

for orientation determination will be validated against direct techniques 

based on x-ray absorption. Models for the quantitative separation of the 

buried and free interfaces of ultrathin films via nonlinear optics will be 

developed and extended to higher order processes such as coherent antistokes Raman scattering. 

Transport Coefficients and Molecular Dynamics 

R.D. Mountain (838) 

CSTL Program: Chemical and Allied Products

Molecular-level simulation of fluids has been identified as an enabling 

technology for the efficient determination of physical properties of fluids in 

conditions where the experiments are expensive and/or hazardous due to 

toxicity, flammability etc. In order for industrial modelers to realize the 

potential benefits of molecular simulations, two (at least) issues must be 

addressed. The first is the need for physically accurate representation of the 

interactions between the molecules of the fluid and the second is the need to 

develop more efficient simulation methods than are currently available. This 

work addresses the second of these issues. 

While it has been possible for decades to use molecular simulations to estimate the shear viscosity and the thermal conductivity of a model fluid, the computational resources required have been too large to move it from a research to an 

industrial setting. Recently, some novel methods for estimating these transport coefficients from simulations have been 

reported, but no systematic evaluation of the merits of these methods has been produced. The objective of this project is 

to determine the computational efficiency and accuracy of these methods compared with existing simulation methods. 

This project is part of a larger effort in the Physical and Chemical 

Properties Division to improve molecular simulation methodology to 

the point where industrial property modelers will be comfortable adding these techniques to their toolkit because it will expand their ability 

to cope with fluids and state conditions where existing methods fail. 

In order to concentrate on 

the second issue, namely 

accurate and efficient determination of transport coefficients, this project focused 

on the Lennard-Jones fluid, a 

widely studied model system 

sometimes called the “fruit fly” of simulations. There are sufficient 

prior results available so that both accuracy and efficiency can be 

evaluated. Three unconventional methods were examined and one, 

Reversed Perturbation Nonequilibrium Molecular Dynamics, was 

found to be competitive with existing methods. This scheme is being 

further examined to see if the computational efficiency can be improved by reducing the system size without compromising accuracy. 

Future work will be performed 

within the context of the FY05 NIST 

Competence Program on Metrology 

to Enable the Realization of Organic 

Electronics Devices. 

An interagency report, NISTIR 

7170 has been issued and an archival publication on system size 

effects is in the early stages of 

preparation.

CSTL scientist addresses 

the need for more efficient 

simulation methods for industrial modelers. 

The averaged time correlation functions (solid line) and 

the time integral for the shear 

viscosity (dotted line) are 

shown in the graph 
159

Ab Initio Mass Spectrometry 

K.K. Irikura (838) 

CSTL Program: Measurement Science for Future Standards and Technology

Traditional electron-impact (EI) mass spectrometry (MS) is used for 

detecting and identifying compounds in the vapor phase. Important 

applications include detecting toxic compounds, verifying compliance with 

international chemical weapons treaties, detecting chemical explosives, 

detecting illegal drugs, and laboratory analysis of organic compounds. ing illegal drugs, and laboratory analysis of organic compounds. Compounds are identified by comparing observed 

spectra with those of candidate compounds, which must be previously known (e.g., in the NIST Mass Spectral Database). Spectra cannot currently be predicted theoretically. However, theoretical developments at NIST over the past 

several years offer new opportunities for developing theoretical means for predicting EIMS. We would like to develop 

a predictive theory, which would make it possible to positively identify compounds not found in the standard databases. 

Another type of mass spectrometry, tandem mass spectrometry (MS/MS) is 

the ionic equivalent of gas-phase pyrolysis. The standard procedure in proteomics research involves protein digestion followed by MS/MS analysis of 

the resulting peptide fragments. Sequence information is obtained from the 

spectra by using a few simple, empirical rules. However, about half the information in the spectra is discarded because it does not conform to the known 

rules. Recent computational chemistry techniques, developed in CSTL under 

an earlier Exploratory Research project, suggest how additional rules might be discovered theoretically. We are using these and other strategies to seek new rules, which will 

increase the power of MS/MS for proteomics applications. This will increase the speed 

and reduce the cost of protein sequencing. 

Experts in CSTL conclude that MS/MS is a higher priority than EIMS. Fortunately, 

MS/MS is also the simpler chemical process from a theoretical perspective. A new NRC 

postdoctoral associate has explored the peptide fragmentation problem by examining 

certain general trends (e.g., the “glycine effect”) that have been observed in large data 

sets. Tripeptides have been chosen as the most appropriate model for the fragmentation 

of tryptic peptides. The goal is to understand the trends and to make them both quantitative and predictive, thus defining new fragmentation rules. Experimental measurements 

will be done to support the calculations. 

Invention of a New Class of Ultra-Fast, Ultra-Sensitive Mass Spectrometers for Kinetics, Reference 

Mass Spectrometry, and Homeland Defense Applications 

J. W. Hudgens (838) 

CSTL Program: Measurement Science for Future Standards and Technology

The project was designed to demonstrate the feasibility for improved mass 

spectrometers based on Hadamard-transform data acquisition. Hadamard 

mathematics describes the most accurate procedures for measuring properties 

of any ensemble. To the chemist, its most familiar form includes a digital representation of the fast-Fourier transform used in modern infrared spectrometers. A goal is to show that Hadamard MS/MS instruments are feasible and 

can rapidly resolve the chemical structures of components in a complex mixture. Hadamard mathematics suggest that such instruments can acquire comprehensive structure determination data of chemical unknowns as much as 

3,000 to 600,000 times faster than current art. 

The first Hadamard time-of-flight (TOF) mass spectrometer incorporating an electron-impact ionizer has been invented, 

and constructed at NIST. The instrument appears similar to a commercial reflection TOF mass spectrometer; however, 

it contains a specialized electron gun, ion optics, and high-voltage electronics that are optimized for high-frequency 

modulation. Laboratory tests have demonstrated that this ionizer can switch ion streams on and off with a 5 ns rise-fall 

CSTL researcher seeks to develop predictive theory for 

analytical mass spectrometry.

MS/MS has gained prominence 

recently as a key technology of 

proteomics. 

CSTL researcher builds a prototype Hadamard time-offlight mass spectrometer (HTTOFMS), measures its performance, and uses test data to 

model a Hadamard-transform 

MS/MS instrument. 

Example of protonated 

tripeptide structure 
160

time when modulated at 13 MHz. These performance specifications will permit Hadamard operation. Integration of this 

hardware with the necessary software is in progress. 

Conventional TOFMS ion optics accelerate individual 

ion bunches down a flight tube to a detector. The detector signal is sent to a 1 GHz multi-channel scalar that 

counts the ions and measures their arrival times. The 

TOF spectrum is simply the graph of ion counts vs. 

time. Because a conventional TOFMS instrument flies 

each ion bunch individually, it has a duty cycle of only 

≈ 2 %. When operating in Hadamard mode, the ionizer 

modulates the ion stream with a simplex code sequence that is derived from a Hadamard matrix. The simplex matrix is 

composed of rows of 1’s and 0’s that instruct the ionizer to switch the ion current on and off. With a Hadamard modulation frequency of 13 MHz, ≈ 500 distinct ion packets are simultaneously in flight. The signal from an HT-TOFMS initially looks like random noise. The mass spectrum is obtained by multiplying this “noise” by the inverse of the simplex 

matrix. The duty cycle of the HT-TOFMS is 50 %. Hence, the HT-TOFMS obtains a 25x to 50x sensitivity improvement from its higher duty-cycle. Other features of Hadamard mathematics further enhance sensitivity and reduce measurement uncertainties. 

The demonstration that any conventional TOFMS can be converted into a more sensitive Hadamard instrument may 

accelerate adoption of this superior technology. Hadamard technology is expected to find application in fields of chemistry such as the study of the rates of chemical reaction or forensic analyses where high sensitivity, sample conservation, 

and high operational speed are essential. If Hadamard principles could be adapted to other variations of MS/MS, say 

those used in the drug discovery process, it would enable the acquisition of complete MS/MS data sets within the duration of a single high performance liquid chromatography (HPLC) peak. Currently, such data campaigns take days to 

execute and can require use of an expensive (≈$1M) ion cyclotron resonance MS in order to conserve sparse samples. 

The present Hadamard mass spectrometer will be used to obtain feasibility data for an MS/MS instrument. The design 

target is an instrument that can measure the kinetics of complex hydrocarbon mixtures. 

Algorithms that reduce the effects of mask errors (which in mass spectrometers arise from non-ideal ion current modulation) on the measurement 

quality are an active research area in mathematics. The community needs 

data from a well-characterized experiment to verify assumptions about the 

nature of Hadamard measurements. This data may facilitate improvements 

in other Hadamard application fields including microscopy, nuclear magnetic resonance (NMR), and medical magnetic resonance imaging (MRI). 

Sonic Nozzles as Primary Standards 

J. D. Wright and A. N. Johnson (836) 

CSTL Program: Industrial and Analytical Instruments and Services

Critical flow venturis, often termed sonic nozzles, are widely used as 

gas flow standards by industry, are recognized as perhaps the most 

precise gas flow meters available today, and are the basis for metering gas flows over a very large range. Prior studies at the NIST flow 

laboratory as well as other flow laboratories, have shown that sonic 

nozzles maintain their calibration stability for more than 30 years 

within 0.2%. The ISO-9300 standard documents procedures for calculating flow from nozzles at the 0.5% relative uncertainty level via 

theoretical discharge coefficients. Because the dominant contributor 

to uncertainty in tests of sonic nozzle performance was the primary systems used to calibrate them, recent improvements in NIST primary standards allow much better validation of the theoretical calibration predictions. This will have a 

significant impact of flow measurement accuracy in industries where sonic nozzles are used as primary flow standards. 

Using the approach described here, users could have a sonic nozzle manufactured with a known throat diameter and use 

discharge coefficients obtained from references to calculate flow from the nozzle. The approach extends to flows that 

are too large to measure by conventional primary standard designs. 

Preliminary numeric simulations have been conducted and suggest that useful Hadamard MS/MS 

instruments are possible. The incorporation of test 

results from the present HT-TOFMS will help us 

optimize trial instrument designs and data acquisition procedures. 

Plans include incorporating error 

correction algorithms into the NIST 

HT-TOFMS in order to generate 

benchmark reference data. 

CSTL researchers seek to validate the 

theoretical calibrations of sonic nozzles, without calibration to primary 

standards, to facilitate inexpensive, 

portable, and accurate gas flow measurements in industrial settings. 
161

Calibrations of a set of sonic nozzles have been completed using our new 34 L, 677 L, and 26 m3 PVTt primary gas flow 

standards, having uncertainties ranging from 0.05% to 0.13%. Additionally, the NIST Precision Engineering Division 

made nozzle shape and throat diameter measurements with uncertainty of 1 micrometer. The dimensional and experimental flow data were compared with the results of theoretical models and the agreement was excellent (<0.05%). This 

work validates much of the theoretical discharge coefficient predictions upon which ISO-9300 is based, in addition to 

identifying limitations of the model. It also identifies applications where users must exercise caution when using nozzles 

without flow calibrations. The results also show the importance of using the true nozzle shape, including the ratio of the 

throat radius to the radius of curvature, Ω, in calculating the discharge coefficient from the model. 

In the future we will extend this effort to 

smaller flows and smaller nozzle diameters 

where dimensional metrology capabilities are 

rapidly improving and we have extensive 

nozzle calibration data. We will also study the 

significance of nozzle shape defects, dirt deposited by flow, and unusual gas properties in 

causing departures from the expected flow 

behavior. 

Precision Densimetry for Primary Temperature Metrology 

M.O. McLinden (838) 

CSTL Program: Measurement Science for Future Standards and Technology 

Temperature is among the most important quantities in a vast array of 

applications, yet recent work at NIST and other National Metrology 

Institutes (NMIs) provides strong evidence that the currently accepted 

temperature scale, known as ITS-90, differs from the true thermodynamic temperature by about 11 mK at 505 K. This work has been done 

with acoustic resonators, which are the leading alternative to the traditional constant volume gas thermometer for the 

determination of thermodynamic temperatures. The present work explores a third, independent method and so would 

provide a consistency check on the acoustic results. This is important for any redefinition of the temperature scale. The 

new method is related to gas thermometry but avoids many of the practical difficulties, which have led to its virtual 

abandonment by the NMIs. With a constant-volume gas thermometer, the temperature is derived from the pressure ratio 

of a fixed quantity of gas at some temperature T and a reference temperature, usually the SI defining temperature of 

273.16 K. However, with the densimeter, the pressure is 

held constant, and the density ratio measured at several 

pressures and extrapolated to zero pressure to yield the temperature. 

This work is enabled by a new apparatus in CSTL, which is 

proving to be more accurate than older methods by a factor 

of ten. This densimeter operates on the familiar Archimedes 

This work was designed to address 

biases in the currently accepted international temperature scale (ITS-90). 

This research has included numerical studies 

to determine the optimum working gas and the 

measurement uncertainties needed to obtain a 

thermometer with an uncertainty of a few 

thousanths of a kelvin. Argon is the best gas 

with neon a close second. 

0.64

0.66

0.68

0.70

02468

Argon, run 2

Argon, run 1

Neon

Density Ratio vs Pressure (MPa)

-2.0

-1.0

0.0

1.0

2.0

0o

30o

60o

90o

120o

150o

180o

210o

240o

270o

300o

330o

2 .0 1 

0 .0

1 .0

2 .0

-2.0

-1.0

0.0

1.0

2.0

0o

30o

60o

90o

120o

150o

180o

210o

240o

270o

300o

330o

2 .0 1 

0 .0

1 .0

2 .0

z/d=-0.401

z/d=-0.297

z/d=-0.193

z/d=-0.09

z/d=0.014

Deviation from circularity (in μm) for 

a 4.83 mm nozzle plotted at 30 degree 

increments at five cross-sections, beginning at the nozzle throat and stepping upstream toward the nozzle inlet.
162

(buoyancy) principle and provides an absolute determination of density. Several twists on the conventional Archimedes 

experiment—including a differential weighing with two sinkers and a magnetic suspension coupling to separate the 

balance from the fluid being measured—reduce the uncertainties and allow operation over a wide range of temperature 

and pressure. For this project, experiments over the range of 234 K to 505 K with argon, nitrogen, and neon as the 

working gas have been carried out. Experimental protocols and data analysis have been demonstrated. Systematic errors 

in the magnetic suspension coupling were found, but, while not small enough to ignore, they were reproducible at the 

level of a few micrograms. While these experiments have demonstrated the feasibility of the method, the present densimeter has uncertainties that are too high for temperature metrology—in particular, the uncertainty in the sinker volumes is large. The method was inverted and used with the measured data to determine, in-situ, the sinker volumes as a 

function of temperature. This reduced the uncertainties in the sinker volumes, and thus the uncertainties of fluid densities measured with this apparatus, by a factor of four compared to values computed using literature values of thermal 

expansivity. Further development of this method into a temperature standard would require a significant effort involving temperature, pressure, density, length, and mass metrologists, but no insurmountable obstacles are forseen. 

Using Flow Cytometry to Assess the Utility of the Biomarker CD20 for the Diagnosis of B-Cell Chronic 

Lymphocytic Leukemia 

L. Wang (831), A. Gaigalas (831), G. Marti (FDA), F. Abbasi (FDA) 

CSTL Program(s): Health and Medical Technologies, Forensics and Homeland Security

Also Supports: Biomaterials, Pharmaceuticals and Biomanufacturing

B-cell chronic lymphocytic leukemia (B-CLL) is 

the most common hematologic malignancy in 

adults and accounts for 30% of all leukemias. 

Flow cytometry has been used to establish the phenotyping profile (identity) of B-CLL by detecting a 

variety of surface antigens CD5, CD19, CD20, and 

CD23 (note: CD stands for “cluster of differentiation”). It has been found that fewer CD20 cell receptors are present in B-CLL patients than in the 

normal state; this is referred to as “down regulation.” However, the number of CD20 cell receptors reported in the literature for the disease state 

varies by more than one order of magnitude. Because of this, the potential biomarker CD20 has 

limited use for the diagnosis of B-CLL. 

This research team has assessed the utility of CD20 

as a critical biomarker for the diagnosis of B-CLL. 

We found that signals from lymphocytes stained 

with CD20 that had been labeled with either fluorescein isothiocyanate (FITC) or with Rphycoerythrin (PE) were lower in B-CLL patients 

than in healthy blood donors. This finding required 

the use of carefully designed and executed control 

experiments. The fluorescence measurements were 

performed on lymphocytes stained with CD20 

FITC, the MESF (Molecules of Equivalent Soluble 

Fluorophore) values were assigned to the stained 

lymphocytes based on calibration curves. The 

down regulation of CD20 in B-CLL patients was 

observed by comparison with values obtained from 

normal donors. Because PE has a much larger 

fluorescence signal than fluorescein, quantifying 

CD20 expression using CD20 with PE may give a 

tighter distribution and a smaller coefficient of 

variation and more accurate results. The work using PE as the fluorophore is in progress. 

NIST/CSTL and FDA researchers work to 

reduce CD20 cell measurement uncertainty 

that has hampered research in B-cell chronic 

lympocytic leukemia. 

200 300 400 500 600 700 800 900 1000

1000

10000

100000

1000000

MESF Value

FL1 Mean Channel Number

The figure shows calibration curves of a FACScan 

flow cytometer (BD Biosciences) involving four separate experiments using NIST reference material 

RM 8640. MESF (Molecules of Equivalent Soluble 

Fluorophore) values are shown as a function of the 

fluorescence channel number. 
163

Development of ICP-OES Determination of Phosphorus as a Primary Measurement Tool for 

Quantitation of Plant Deoxyribonucleic Acid (DNA) 

M.J. Holden (831), M. Winchester (839), J.R. Blasic, Jr., M. Dizdar, P. Jaruga, and Y. Tewari (831) 

CSTL Program(s): Measurement Science for Future Standards and Technology

Also supports: Biomaterials, Pharmaceutical and Biomanufacturing, Food and Nutritional Products, Forensics and 

Homeland Security, Health and Medical Technologies

The successful detection and quantitation of biotech crop material in 

grain or food is highly dependent on the acquisition of pure and nondegraded DNA in a quantity that is appropriate for the limits of detection and quantitation of the measurement methods. The most important measurements, related to US export of biotech commodity crops 

and prepared foodstuffs, are the ones relevant to the detection of trace 

amounts of biotech material. Thus the amount of DNA that is used for 

the detection becomes critical. Laboratories currently use spectroscopic methodologies to quantitate DNA preparations, for example, 

DNA absorbance at 260 nm or fluorescent-dye binding. The values 

obtained can be seriously compromised by impurities in the DNA 

preparations or the state of the DNA itself. 

The primary methods developed will support the development of 

Standard Reference Materials for the calibration and validation of 

plant DNA measurements using other methods that are appropriate for 

testing and research laboratories. In this project the primary measurands are phosphorus and the four nucleotide bases that comprise 

DNA. A substantial effort has been directed during FY04 toward the 

development of a high-performance inductively-coupled plasma optical emission spectrometric (HP-ICP-OES) method for determining the 

total mass of phosphorus present within a given sample of DNA. HPICP-OES employs a clever experimental design, a well-chosen internal 

standard, and an innovative drift correction technique to enable expanded uncertainties on the order of a few parts per thousand. A 

methodology for measuring phosphorus has been developed, and several determinations using phosphorus spectrometric solution standards 

as 'mock' DNA samples have been demonstrated. As a more realistic 

test, a sample of corn DNA has been successfully digested, and suitable phosphorus spectra have been acquired. Quantitation of real 

DNA samples using the HP-ICP-OES approach is forthcoming. 

A remaining challenge concerns instabilities within the microconcentric nebulizer that is required for the combination of 

high sensitivity and small sample volumes. High Performance Liquid Chromatography (HPLC) is a second unrelated 

technique for the quantitation of phosphorus. The team has developed a suitable digestion protocol to release phosphorus and measured the phosphorus mass in DNA preparations. This methodology will provide an independent validation 

of the phosphorus content. Nucleotide analysis is the other critical component. This analysis is accomplished using 

both gas chromatography and liquid chromatography coupled with mass spectroscopy. These investigations have 

shown that DNA from corn kernels and soybeans responds differently to the digestion and analysis protocols that work 

with human DNA. Work is continuing to find the best protocol suitable for use with plant DNA. 

CSTL researchers develop primary methods that provide accurate and traceable measurement of 

total plant DNA. 

DNA quantitation plays an important 

role in commerce, for example it provides a basis for the detection of biotech crop material commingled with 

conventional crops. 

The importance of this work has been highlighted recently in experiments we conducted to compare dye 

binding properties of plant DNA with that of animals and microbes. Significant differences in response 

were observed with two commonly used fluorescent dyes which highlight the inappropriate use of mammalian and microbial DNA as calibrants for plant DNA measurements and the necessity of new plant 

DNA Standard Reference Materials. 
164

Chiral Temperature Gradient Focusing 

D. J. Ross (839), K. Balss (836), K. Phinney, and W.N. Vreeland (839) 

CSTL Program: Measurement Science for Future Standards and Technology 

Temperature gradient focusing (TGF) was demonstrated for the 

simultaneous concentration and separation of the enantiomers 

of chiral molecules. The new technique was found to provide 

high performance in a number of areas desirable for chiral separations including rapid separation optimization and method development, facile peak order reversal (desirable for enantiomeric purity measurements), and high resolving power (comparable to capillary electrophoresis) in combination with greater 

than 1000-fold concentration enhancement for improved detection limits. In addition, chiral temperature gradient focusing was shown to offer a capability for real-time monitoring of the interaction of chiral analyte molecules with chiral 

selectors that could potentially be applied to the study of other molecular interactions.

The major accomplishment of the last fiscal year was to demonstrate that chiral TGF works using cyclodextrins as 

chiral selectors for the concentration and separation of chiral amino acids and pharmaceutical molecules. In addition, it 

was shown that this new technique can provide performance as good as and in some aspects superior to that provided by 

conventional techniques. Specifically: 

• Greater than 1000-fold concentration enhancement of chiral molecules was demonstrated. 

• Chiral TGF was shown to provide resolution comparable to capillary electrophoresis (the best available conventional technique). 

• Method optimization was found to be very rapid with chiral TGF because the same focused sample could be 

retained and held focused in the separation channel while different temperature gradients, electric field 

strengths, different chiral selectors, or chiral selector concentrations were tested. 

• Peak order reversal was also found 

to be very easy with chiral TGF. 

Simply by reversing the direction of 

the applied temperature gradient and 

the polarity of the applied voltage, 

the order of the focused enantiomer 

peaks could be reversed, facilitating 

the analysis of small amounts of impurity enantiomer in a nearly enantiomerically pure sample as shown in 

the figure to the right. 

• It was also found that chiral TGF of 

chiral drugs in urine could be successfully performed with no sample 

preparation. 

Chiral TGF separations can be performed in much shorter and simpler 

microfluidic channels or capillaries 

than conventional separation techniques such as HPLC and CE. Because of this, chiral TGF, and TGF 

in general, should be much more 

compatible with miniaturization and 

integration into microfluidic, lab-ona-chip systems.

CSTL researchers develop and demonstrate 

electrokinetic focusing methods for the 

high-performance separation of chiral 

molecules with an emphasis on molecules of 

biochemical and pharmaceutical interest. 

a

0

20

40

60

80

0 0.5 1 1.5 2

c

Distance (mm)

Intensity (AU)

b

0

20

40

0 0.5 1 1.5 2

Distance (mm)

Intensity (AU)

d

Trace analysis of enantiomeric purity by chiral TGF. (a) Dansyl-lglutamic acid with a mole fraction of 0.05% dansyl-d-glu. Focusing conditions: T1 = 10 °C, T2 = 30 °C, +1000 V/cm (see Fig. 1A), 

1 mol/L Tris-borate with 20 mmol/L g-CD. (b) Dansyl-d-glu with 

a mole fraction of 0.1% dansyl-l-glu. Focusing conditions: T1 = 40 

°C, T2 = 10 °C, -1000 V/cm, 1 mol/L Tris-borate with 20 mmol/L 

g-CD. (c) Intensity vs. distance plots for (a, red curve) and similar 

results with no impurity (blue curve). (d) Intensity vs. distance 

plots for (b, red curve) and similar results with no impurity (blue 

curve). For clarity, the red curves in (c,d) have been offset. 
165

Determination of Low Level Nitrogen by Radiochemical Neutron Activation Analysis 

R.L. Paul and R.M. Lindstrom (839) 

CSTL Program: Industrial and Analytical Instruments and Services

Many alterations of metal and semiconductors properties are ascribed to the 

presence of low-level nitrogen. Current industrial methods for measurement of 

nitrogen are not reliable below 1 mg/kg. CSTL researchers’ expertise in radiochemical neutron activation analysis (RNAA) was exploited to develop a 

method to detect nanogram levels of nitrogen in materials. In this procedure, 

nitrogen is measured via the 14N(n, p)14C reaction with thermal reactor neutrons, 

and the 14C formed is separated and quantified by beta counting. The method is 

matrix independent and chemically specific, and since detection is based on a 

nuclear rather than a chemical reaction, the results are independent of the 

chemical form of nitrogen. Furthermore, since only radioactive 14C is being 

quantified, the blank problems inherent in non-nuclear methods for nitrogen 

analysis are avoided. 

Several alternative procedures for oxidation of the steel have been tried including oxidation by fusion with Pb3O4/B2O3

in an enclosed system under a flow of argon. However this is both lengthy and expensive, and some unreacted steel remained even after an hour at 950 °C. In addition, there is no way to determine the fraction of 14C recovered from the 

sample. However, the most promising method of attack is simple combustion in flowing oxygen at 900 °C to 1000 °C, 

with or without an accelerator, a method that has been used in the analysis of carbon in steel for many years. This 

proved to work well and was utilized in the procedure developed for low alloy steels and is described below. 

The steel sample is mixed with a known amount of nonradioactive 

carrier (CaCO3), before oxidation. The evolved CO2 is precipitated 

as BaCO3, which is then weighed to determine the chemical yield 

and decomposed with HCl. Evolved CO2 is quantitatively absorbed in the scintillator cocktail. Tests have verified that the fraction of BaCO3 recovered is near 100 %. A known quantity of a 14C-labeled BaCO3 was decomposed with HCl, and 14CO2 absorbed 

in scintillation cocktail. Repeated trials yielded consistent and 

quantitative results for low alloy steels. Preliminary measurements 

indicate that the proposed scheme has a high probability of success. 

This scheme will be tested first by burning samples of un-irradiated 

steel mixed with CaCO3 carrier and a known amount of 14C tracer. 

If the 14C can be quantitatively measured, then the method will be 

further tested through analysis of several NIST SRM steels that are 

certified for nitrogen. 

Studying the Chemistry of Pharmaceuticals in Wastewater 

M. Bedner and W.A. MacCrehan (839) 

CSTL Program: Energy and Environmental Technologies 

Pharmaceuticals and personal care products (PPCPs) are increasingly being recognized as a threat to human health and the environment. PPCPs are continuously released into the environment 

from human and agricultural waste and are frequently subject to 

waste treatment processes. Chlorination is the most widely-used 

chemical process for disinfecting wastewater and drinking water. 

Chlorine is a strong, non-selective oxidant that is capable of rapidly transforming pharmaceutical compounds. Understanding the 

chemical and toxicological nature of the transformation products is 

an important first step to determining what compounds should be 

measured in the environment. 

A quantified radiochemical 

neutron activation procedure that uses liquid scintillation counting was developed to measure lowlevel nitrogen in steels and 

other materials to meet a 

critical industrial need. 

Results: A reliable method for measurement of nitrogen in a wide variety of materials could impact many industries. 

Foremost on this list is the US aerospace 

industry, which requires primary standards for accurate measurement of lowlevel nitrogen in titanium. The RNAA 

method could provide the industry with 

the means to improve safety standards for 

aircraft. The method could be extended to 

analysis of other materials, especially 

biologicals, where determination of nitrogen in proteins is essential. 

Preliminary investigations conducted by 

CSTL researchers provide critical information to be used by environmental 

researchers determining the importance 

and fate of pharmaceutical compounds. 
166

The identification of unknown transformation products using mass spectrometric detection is critical in investigating 

this measurement problem. Four pharmaceutical compounds that are frequently detected in the environment were evaluated and included; acetaminophen, metoprolol, sulfamethoxazole, and diclofenac. We have begun investigations of the 

pharmaceutical transformations using calibrated doses of chlorine (added as hypochlorite). Liquid 

chromatography was used to separate reaction products, which were evaluated and identified using 

four detection modes. All compounds were significantly transformed by reaction with chlorine. 

Acetaminophen was shown to form two toxic products, benzoquinone and N-acetyl-pbenzoquinoneimine, and at least ten additional products (see chromatogram). Metoprolol reacted 

to form one major product, a chloramine. Sulfamethoxazole formed two major products, both of 

which have a chlorine atom in the molecule, but only one of which appears to be a chloramine. 

Diclofenac forms at least five products, one of which has an additional chlorine atom. For all 

compounds, the products of the reactions tended to be more hydrophobic than the parent pharmaceutical, which might 

make them more bioaccumulative. 

This project is still in its early stages, so there are 

many important aspects that need to be addressed. 

The transformation rates of the pharmaceutical compounds need to be evaluated at the ng/g levels typically found in wastewater to verify that the reactions 

will be significant. Also, as products are identified, 

targets for measurement in the environment need to 

be determined based on the potential for toxicity 

and/or bioactivity. In addition, there are other 

chemical processes including chloramination, 

dechlorination, and ozone-disinfection that might 

transform pharmaceutical compounds. The effects 

of these water treatment processes on pharmaceuticals will be investigated. 

High-Throughput Bioimaging with Quantum Dots: HER2 Testing Standard 

Yan Xiao and Peter E. Barker (831) 

Details provided in CSTL Program: Health and Medical Technologies

New HIV Bioinformatics Database 

T. N. Bhat(831) and A. Wlodawer (NCI, NIH) 

Details provided in CSTL Program: Health and Medical Technologies

“Structure-Based Browsing and Retrieval” aspects of the work was supported by Exploratory Research

Computational Chemistry Illuminates Atomistic Processes at Complex Interfaces 

A.M. Chaka (838) 

Details provided in CSTL Program: Chemical and Allied Products

Acetaminophen

Two toxic 

products

Over 10 additional products 

of unknown toxicity

0

5

10

15

20

25

0 5 10 15 Separated Components

Signal Intensity

Acetaminophen

Two toxic 

products

Over 10 additional products 

of unknown toxicity

0

5

10

15

20

25

0 5 10 15 Separated Components

Signal Intensity

Structure of 

acetaminophen 
167

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 2004, the following staff members were formally recognized for their achievements. 

Steven Choquette (839), Edgar Etz (837), Wilbur Hurst (836), and Douglas 

Blackburn (839) received the Edward Bennett Rosa Award for the development 

of SRM 2241 through SRM 2244, Raman intensity correction standards that will 

enable U.S. industry to accurately inter-compare data. 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 either within NIST or in cooperation with other 

Government agencies or private groups. 

Rusty Day (839) received the Conference of Southern Graduate Schools 

(CSGS), Masters Thesis Award. 

Colleen Bryan (839) received the Best Student Poster Award for “Non-Lethal 

Monitoring of Trace Elements in Bottlenose Dolphins, Tursiops truncatus” at the 

Southeast and Mid-Atlantic Mammal Symposium.

Michael Frenkel, Robert Chirico, and Qian Dong (838) 

received the US Department of Commerce Bronze Medal for 

their pioneering efforts in developing and promoting 

widespread acceptance of an important new communication 

process for thermodynamic data 

R. F. Hafer and A. Laesecke (838) received the Best Paper Award by the Journal Measurement 

Science and Technology for their paper “Extension of the Torsional Crystal Viscometer to 

Measurements in the Time Domain,” Meas. Sci. Technol., 14, 6633-6673 (2003). 
168

Marcia Holden (831) received, in conjunction with the USDA Grain Inspection, Packers and 

Stockyards Administration Biotechnology Team, the 2004 USDA Group Honor Award for 

Excellence. This award was conferred for developing and implementing programs that have 

enhanced economic opportunities for United States producers and the grain industry to provide 

domestic and international confidence in United States grain. 

Donna Kimball (839) received the CSTL Secretarial Achievement Award for outstanding skills 

and providing extraordinary service to the staff and programs of CSTL. 

Patrick Looney (836) was elected a Fellow of the American Vacuum Society (AVS) in recognition 

to his many contributions to advances in vacuum measurements technologies related to gauging and 

partial pressure analysis 

Gary Mallard and Peter Linstrom (838) received the Judson C. French Award for their 

development of the NIST Chemistry WebBook, which has revolutionized the delivery of 

physical and chemical property data to NIST customers. The Judson C. French Award is 

granted for significant improvement in products delivered directly to industry, including 

new or improved NIST calibration services, Standard Reference Materials, and Standard 

Reference Databases. 

Dave Matheu (838) received the Richard A. Glenn Award from the American Chemical Society 

(ACS) Fuel Division for his paper entitled “Automated Construction of Chemical Mechanisms for 

Challenging Pyrolysis Systems/Pressure-Dependent Automated Mechanism Generation”. 

George Rhoderick (839) received the US Department of Commerce Bronze Medal for his 

outstanding efforts in the development of a series of Volatile Organic Compound (VOC) in Air 

standards.
169

Dean Ripple and Gregory Strouse (836) received the US Department of 

Commerce Bronze Medal for determining the errors in the ITS-90 within the 

industrially significant range from 273K to 505K and forming the basis for future 

temperature scale. 

Dean Ripple (836) received the ASTM Award of Merit from Committee E-20 (Thermometry). 

David Simons (837) was elected a Fellow of the American Vacuum Society (AVS) in recognition 

of his development of ion implanted Standard Reference Materials for advanced semiconductor 

metrology. 

Mark Sobolewski (836) received the US Department of Commerce Bronze Medal for 

development of unique electrical measurement methods and mathematical models used in etching 

plasmas widely used in semiconductor manufacturing. 

Michael Tarlov (836) received the US Department of Commerce Silver Medal for his seminal and 

outstanding contributions to the science and technology of self-assembled monolayers. 

Cynthia Zeissler (837) received the US Department of Commerce Bronze Medal for 

extraordinary innovation in developing and applying spatially resolved radiation detection 

methods for homeland security and national defense.
170
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, at the Center for Advanced Research in Biotechnology 

(CARB) in Rockville, MD, and at the Hollings Marine Laboratory (HML) in Charleston, SC. 

Chemical Science and Technology Laboratory

Dr. Willie E. May, Director 

(301) 975-8300 

willie.may@nist.gov 

Dr. William F. Koch, Deputy Director 

(301) 975-8301 

william.koch@nist.gov 

Biotechnology Division

Dr. Robert N. Goldberg, Acting Chief 

(301) 975-2584 

robert.goldberg@nist.gov 

3⁄4 DNA Technologies 

3⁄4 Bioprocess Measurements 

3⁄4 Structural Biology 

3⁄4 Cell and Tissue Measurements 

Process Measurements Division

Dr. James R. Whetstone, Chief 

(301) 975-2600 

james.whetstone@nist.gov 

3⁄4 Fluid Flow

3⁄4 Process Sensing 

3⁄4 Thermometry

3⁄4 Pressure and Vacuum

3⁄4 Thermal and Reactive Processes

3⁄4 Fluid Science

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 TRC Group 

3⁄4 Chemical Reference Data 

3⁄4 Computational Chemistry 

3⁄4 Experimental Properties of Fluids 

3⁄4 Theory and Modeling of Fluids 

3⁄4 Cryogenic Technologies 

Analytical Chemistry Division

Dr. Stephen A. Wise, Chief 

(301) 975-3112 

stephen.wise@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 2005