Chemical Science and Technology Laboratory 

Annual Report – FY2002 

NISTIR 6954 
NISTIR 6954 

Chemical Science and Technology Laboratory 

Annual Report - FY2002 

Hratch G. Semerjian, Director 

Chemical Science and Technology Laboratory

February 2003 

U. S. DEPARTMENT OF COMMERCE 

Donald L. Evans, Secretary

TECHNOLOGY ADMINISTRATION 

Philip J. Bond, Under Secretary for Technology

NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY 

Arden L. Bement, Jr., Director
Compiled and Edited by Hratch G. Semerjian, William F. Koch, Ellyn S. Beary, and Karen 

Phinney. Cover design by Ann 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 2003 
 

 

 

Table of Contents 

 

Page 

Message from the CSTL Director iii 

A. CSTL Overview 1

 

B. CSTL Programs 

 

1. Automotive and Aerospace 15 

2. Biomaterials 19 

3. Pharmaceuticals and Biomanufacturing 23 

4. Chemical and Allied Products 29 

5. Energy Systems 45 

6. Environmental Technologies 51 

7. Food and Nutritional Products 67 

8. Forensics and Homeland Security 71 

9. Health and Medical Technologies 87 

 10. Industrial and Analytical Instruments and Services 99 

 11. Microelectronics 117 

 12. Measurement Standards 127 

 13. Data and Informatics 141 

 14. Technologies for Future Measurements and Standards 145 

C. Exploratory Research 155 

D. Staff Recognition 169 

i
ii
Chemical Science and Technology Laboratory

The year 2002 marked the tenth year of my tenure as the Director of the 

Chemical Science and Technology Laboratory at NIST. It has been a very 

fulfilling period of my career; I have had the pleasure and honor to lead our 

outstanding research staff in transforming CSTL into a high-impact, 

customer-focused organization. CSTL is recognized as the world’s leading 

chemical metrology laboratory; our prestige in the international 

measurements and standards community has created increased demands for

our staff to provide leadership for many international comparisons, symposia, and training

activities. During the last few years of flat budgets, it has become ever more important to articulate the economic impact of our work, and to focus our efforts on projects and programs that will

create the largest return for the nation. Our continuing interactions with industry to help us prioritize our work have been critical, and we all appreciate the ideas and input our industrial colleagues have shared with us so generously. 

The last two years have also represented a time of great changes in technology and national 

priorities, in the aftermath of September 11th. NIST’s (and NBS’s) illustrious hundred-year history, 

its technical excellence, broad range of capabilities and flexibility has served us well towards responding to the changing needs of the nation. While maintaining our focus on core mission responsibilities of measurement science and standards, NIST and CSTL are expanding our programs on Nanotechnology, Health and Medical Technologies, Data and Informatics, and Homeland Security. The main body of this report reflects these changes in our programs. 

Today’s Chemical Science and Technology Laboratory has the most comprehensive array of

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

science and technology. We are well positioned to achieve our Vision to be a world-class research laboratory that is recognized by the nation as the primary source for the chemical, biochemical, and chemical engineering measurements, data, models, and reference standards that

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

Finally, I write these remarks with a heavy heart, thinking about the recent and tragic loss of the

crew of the Space Shuttle Columbia. NIST has had several experiments on board space shuttles

over the past years, and had one on the ill-fated Columbia. Experiments in the microgravity of 

space remove limitations experienced here on earth. Ironically, the crew of the Columbia had 

transmitted previously unattainable data back to earth during its mission, completing a highly successful experiment on our behalf. We hope that our use and dissemination of the data they provided will be a tribute to their lives, and a fitting contribution to their legacy. 

Hratch G. Semerjian, Director 

Chemical Science and Technology Laboratory 

iii
http://www.nist.gov

Arden L. Bement, Jr., Director 

Karen H. Brown, Deputy Director 

iv

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The National Institute of Standards and Technology is a non-regulatory 

federal agency within the U.S. Commerce Department's Technology

Administration. Founded in 1901, NIST's mission is to develop and

promote measurements, standards, and technology to enhance

productivity, facilitate trade, and improve the quality of life. NIST has an

operating budget of about $810 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. 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 recognizes business performance excellence and quality

achievement by U.S. manufacturers, service companies, educational organizations, and 

health care providers. The Malcolm Baldrige National Quality Award, created in 1987, is 

widely credited with making quality a national priority. 
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, 

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

equity in trade, and improving public health, safety, and environmental quality. With current world 

events, the need for accurate and timely chemical and biological measurements is more imperative than ever. CSTL is ready to meet these new challenges. 

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

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

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

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

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

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

worldwide working in chemical science and technology. 

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

that is recognized by the nation as the primary source for 

the chemical, biochemical, and chemical engineering 

measurements, data, models, and reference standards 

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

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

Laboratory providing the chemical measurement infrastructure to: 

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

• assure equity in trade; and 

• improve public health, safety, and environmental 

quality. 

Director 

 Hratch G. Semerjian 

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

CSTL has operated under a published Strategic Plan for more than a decade. In the context of 

this plan CSTL developed its mission, vision and goals. These are clearly aligned with the current 

NIST 2010 Strategic Plan. 

Periodically, CSTL’s Strategic Plan is reassessed and updated to respond to National priorities, 

changing customer needs, and the rapid evolution of science and technology. Although the details of the strategic planning process have varied over the years, its essential characteristics — 

customer-focused and open to all stakeholders — have been preserved. CSTL’s Strategic 

Plan focuses on achievement of three strategic goals: 

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

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

objective is achieved by: 

• Developing and demonstrating international comparability for chemical and physical 

measurements, 

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

• Supporting and strengthening voluntary standards organizations.

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

and processes. This objective is realized by: 

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

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

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

and processes; and 

• Developing procedures or protocols for data access, facilitating data exchange, and by disseminating properly 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 or advanced technology development and dissemination. 
3

Organizational Structure and Resources 

CSTL is organized to reflect the technical expertise that is the foundation of our technical programs and allows us to accomplish our mission. The Laboratory consists of five Divisions: Biotechnology Division, Process Measurements Division, Surface and Microanalysis Science 

Division, Physical and Chemical Properties Division, and Analytical Chemistry Division. Each 

Division employs a group structure organized to achieve synergy and critical mass in its technical 

program areas. 

In FY2002, Dr. Gary Gilliland stepped down as Chief of the Biotechnology Division and has 

assumed the role of Associate Director of CARB. Dr. Vincent Vilker took on the job of Chief of 

the Biotechnology Division. Dr. W. Mickey Haynes retired as Chief of the Physical and Chemical 

Properties Division on January 3, 2003. Dr. Gregory Rosasco, retired Chief of the Process 

Measurements Division, returned to active duty to replace Mickey. The other significant change 

in CSTL’s organization is reorganization in the Surface and Microanalysis Science Division, 

resulting in the reassignment of all staff in the Atmospheric Chemistry Group to other appropriate 

groups in the Division, more in keeping with the disciplinary nature of our groups and divisions. 

To achieve its goals, CSTL maintains an experienced, well-educated professional staff. The fulltime permanent staff numbered 273 in FY02, in addition to 83 temporary and part-time employees. Also, there were 237 guest researchers working closely with CSTL staff in various aspects of 

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

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

augments the professional staff. Approximately 75% of the technical staff have PhD degrees. 
4

Administrative 

Support (37) 

Technical 

Support (33) 

Chemists 

(147) 

Physicists 

(44) 

Engineers, 

 Comp. Scientists 

(40) 

Biologists, 

Biochemists (30) 

Students (25)

CSTL Staff, FY 2002 CSTLFunding,FY 2002

Other Agency 

19% 

Competence 

5% 

ATP 4% 

Research Base

59% 

 Measurement Services

13% Funding Sources

Total = $64.6M

CSTL’s physical facilities are located at the major NIST sites in Gaithersburg, Maryland and 

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

Rockville, Maryland and the Hollings 

Marine Laboratory (HML) in Charleston, South Carolina. The Physical 

and Chemical Properties Division 

has operations located at the NIST 

sites in Gaithersburg and Boulder. 

Biotechnology Division staff work 

closely with that of the University of 

Maryland Biotechnology Institute 

located at CARB. The Analytical 

Chemistry Division is the primary 

liaison with HML, having staff located in Charleston. HML is a cooperative research facility involving 

NIST, the National Oceanic and Atmospheric Administration, the South Carolina Department of 

Natural Resources, the University of Charleston, and 

the Medical University of South Carolina. 

The Analytical Chemistry and Biotechnology Divisions 

have now been located in the Advanced Chemical Sciences Laboratory (ACSL) for over three years, the first 

new research facility on the NIST Gaithersburg Campus in over thirty years. Although ACSL adds significantly to CSTL’s research capabilities, it does not meet 

all facility needs of our program. 

NIST is proceeding with implementing its facilities 

master plan, the current focus of which is the Advanced Measurements Laboratory (AML). Construction of the Advanced Measurements Laboratory 

(AML) on the Gaithersburg campus is well underway 

with completion planned for 2004. This new facility 

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

and Temperature activities in the Process MeasureHollings Marine Laboratory 
5

ments Division. Attention is now being directed to improving the facilities for those units not located in the ACSL and AML, including Boulder. 

CSTL Strategic Directions 

As part of its most recent update of the Strategic Plan, 

CSTL has established five Strategic Directions: 

• Biotechnology 

• Nanotechnology 

• Healthcare 

• Data and Informatics 

• International Measurement Standards 

The last one reflects an increased emphasis on issues related to global trade. New projects 

started over the last five years reflect these Strategic Directions. Investment decisions are made 

jointly by the Laboratory Director, Deputy Director and Division Chiefs. CSTL’s decisions are in 

clear alignment with the NIST 2010 Programmatic Strategic Focus Areas (SFAs) shown above (in 

box). Some of the projects selected contribute to more than one Strategic Direction, and some 

contribute to Homeland Security. 

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

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

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

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

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

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

• Industrial Need - Magnitude and immediacy of 

industrial need 

• Match to Mission – CSTL provides the Nation’s 

chemical measurement infrastructure 

• Making a Difference – Is the CSTL contribution 

critical for success? 

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

• Timely Quality Output - The ability to respond in 

a timely fashion with high-quality output 

• Science/Technology Opportunity - Recent scientific and technological advances present new 

opportunities 

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

technologies and measurement methods. In FY2002, CSTL funded 12 such projects, following a 

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

C of this Annual Report. 

NIST Programmatic SFAs 

• Nanotechnology 

• Healthcare 

• Information /Knowledge 

 Management 

• Homeland Security 

NIST Program Selection Criteria 

Include the Following Elements 

• Fit to Mission 

• Unique NIST value-add 

• Potential external impact 

! Customer needs 

! Impact path 

! Strategic significance of 

impact arena 

• Potential impact on NIST

• Probability of success
6

CSTL Programs 

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

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

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

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

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

such as biotechnology. 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. 

CSTL’s programs are customer-based and 

serve to focus our activities. The agility of our 

technical staff permits CSTL to respond in a 

timely manner to changes in customer needs 

within each industry segment, changes in 

priorities among industries as well as National 

priorities. CSTL has identified 11 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. 

It is also beneficial to articulate CSTL projects 

by activities that cross-cut industry sectors. 

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

three strategic goals. 

1. Automotive and Aerospace 

2. Biomaterials 

3. Pharmaceuticals and Biomanufacturing 

4. Chemical and Allied Products 

5. Energy Systems 

6. Environmental Technologies 

7. Food and Nutrition 

8. Forensics and Homeland Security 

9. Health and Medical Technologies 

10. Industrial and Analytical Instruments and 

Services 

11. Microelectronics 

12. Measurement Standards 

13. Data and Informatics 

14. Technologies for Future Measurements 

and Standards 
7

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 

our programs, to assess the quality and relevance 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 direct evidence for the quality of our technical programs. Our performance in CIPM Key 

Comparisons in a broad range of measurement fields, where our performance is compared to the 

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

demonstrate our global leadership. 

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

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

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

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

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

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

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

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

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

customers. 

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

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

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

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

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

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

and improving our prioritization process to reduce out-of-stock items. CSTL is also making special efforts to expand our web-based data dissemination efforts, and to provide more standardized formatting and single-portal access to all CSTL databases. 
8

Some of the CSTL outputs and interactions in FY2002 are summarized in the following table. 

Selected CSTL Outputs – FY 2002

Div. Pubs. 1 Talks Committees2 Seminars Conferences CRADAs Patents 

Issued 

SRMs

/RMs3

SRDs Cals.4

830 5 14 25 11 2 0 0 0 0 0 

831 76 117 65 14 7 1 1 1 1 0 

836 70 90 95 12 5 1 0 1 0 832 

837 46 141 85 4 12 1 0 5 3 0 

838 80 124 97 19 1 7 1 2 10 2 

839 82 157 127 17 11 1 0 73 1 245 

Total 359 643 494 77 38 11 2 82 15 1079 

1 Publications appearing in print in FY2002. Another 188 manuscripts have been submitted for publication. 2

Committee totals include 64 editorships 

3

SRMs/RMs (Certificates issued) 

4

Calibrations were performed for over 338 customers 

Division Key: 

830 Laboratory Office 837 Surface and Microanalysis Science Division 

831 Biotechnology Division 838 Physical and Chemical Properties Division 

836 Process Measurements Division 839 Analytical Chemistry Division 

SRM – Standard Reference Material SRD – Standard Reference Data 

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

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

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

the influence and reach of our programs.

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

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

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

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

the need to measure and analyze current and past performance but also contribute to future strategic planning. Practically, only a few of these in-depth analyses are possible each year. In 

FY2002, we completed a study of our NIST Traceable Reference Materials (NTRM) program, 

described in more detail immediately below. In addition, we summarize some of the results of 

earlier impact studies, with updated information on subsequent activities and benefits accrued 

since the publication of each report. 

NTRM Gas- Mixture Standards: 

The NIST Traceable Reference Materials (NTRM) program was created to help address the problem of increasing needs for reference materials with a well-defined linkage to national standards. 

An NTRM is a commercially produced reference material with a well-defined traceability to existing NIST standards for chemical measurements. This traceability linkage is established via criteria and protocols defined by NIST and tailored to meet the needs of the metrological community 

to be served. The NTRM concept has been implemented in the gas standards area to allow NIST 

to respond to increasing demands for high quality reference materials needed to implement the 

"Emissions Trading" provisions of the Clean Air Act of 1990. 
9

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

Cholesterol Standards: 

Chemical metrology is at the heart of accurate medical 

diagnosis and the development of measures to improve our 

health and ensure long life. In the U.S. more than one trillion 

dollars are spent each year on health care, which is about 14 

% of the U.S. GDP. More than 20 % of these expenditures are 

for measurements. 

Over the last three decades, NIST, in cooperation with the 

College of American Pathologists (CAP), has developed a series of highly accurate and precise 

methods for a number of clinically important serum constituents, including cholesterol. These 

methods are recognized by the international clinical laboratory community as "definitive" and 

have been used to certify a series of cholesterol SRMs. The first pure crystalline cholesterol 

(SRM 911) was introduced in 1967. Using the definitive method, serum cholesterol SRMs were 

developed in 1981 (SRM 909) and again in 1988 (SRMs 1951 and 1952). These SRMs have led 

to a steady decrease in the measurement uncertainty from 18% relative in 1969 to about 6% in 

1999. The economic consequences of NIST’s Cholesterol Standards Program are experienced 

at several levels of the supply chain from manufacturers, to network laboratories, to clinical laboratories that ultimately deliver medical services to the consumer. The results of this study indicate that NIST has played an important economic role in support of a national effort to monitor, 

measure, and control cholesterol levels, thereby contributing to the reduced level of heart disease. The economic impact study estimates a benefit-to-cost ratio of 4.5, and a social rate of return of 154 %. The net present value was calculated to be more than $3.5 million. This report 

was published in September of 2000, and the timeline extended from 1986 to 1999. 

 Recent Developments: NIST Cholesterol SRMs remain 

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

kits. Approximately 500 SRM units were sold per year 

over the past three years. These SRMs also serve as 

important “high order” reference materials, providing 

measurement traceability for in vitro diagnostic (IVD) 

These 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. 
10

products. This level of traceability will soon be required for IVD products to be imported into 

European markets, as specified in the EU IVD Directive set to go into effect in December of 2003. 

Since U.S. based companies supply 60 % of the IVD products sold in Europe, it is critical to U.S. 

industry that NIST maintain and expand SRMs that provide the required traceability. 

Sulfur in Fossil Fuels: 

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

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

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

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

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

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

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

from the mine and well to power plants and refineries requires 

precise and accurate determination of sulfur content in two or more 

laboratories. For equity in trade and the efficient production of 

energy, it is mandatory that instrumentation in these laboratories 

be calibrated using accurate standards. 

NIST has certified the sulfur content in about 30 coal and fuel oil 

SRMs to an accuracy of better than ± 0.1 % relative, using isotope 

dilution mass spectrometry. These SRMs provide industry with the 

primary calibration materials needed for instrumentation used in 

routine measurements. SRMs also provide industry with a strong 

traceability link to NIST for such measurements, whether they be 

for setting the price of fuel or for demonstrating compliance with 

environmental regulations. Surveyed industry representatives 

indicated that NIST SRMs have decreased the level of uncertainty 

associated with their measurements of sulfur content. This reduction has led to economic 

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

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

sulfur emissions to the environment. This study, published in February 2000 estimates a benefitto-cost ratio of 113, and a social rate of return of 1,056 %. The Net Present Value was calculated 

to be more than $400 million. The time period studied was from 1984 to 1999. 

Recent Developments: The petroleum refining industry faces a June 2006 deadline to comply 

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

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

for at least 80% of the highway diesel fuel produced, 

with a 500 μg/g (ppm) cap on the remaining 20% of their 

production. By 2010, all highway diesel fuel must meet a 

15 μg/g (ppm) cap. In addition, the industry expects that 

the EPA will propose similar regulations for the non-road 

diesel fueled 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. NIST is in the 

process of releasing a new diesel fuel SRM with a sulfur 

concentration of 11 μg/g (ppm) that will meet the new 

need of the industry. 

A new twist on existing standards: 

New environmental regulations 

have capped Hg emissions at 26 

tons starting in 2010 and reduced to 

15 tons by 2018. There has been 

value-added to NIST’s eight natural 

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

reanalyzed and certified for Hg. 

These new certifications will take on 

significant importance since they 

will be used increasingly by the 

electric power industry to ensure 

compliance with these new regulations.
11

Alternative Refrigerants: 

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

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

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

research that would allow industry to make the switch to alternative 

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

rules, and then started research on determining the physical properties 

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

through NIST’s Standard Reference Data Program. The 

REFPROP program enables 

manufacturers and users of alternative refrigerants to 

model the behavior of refrigerant mixtures in their respective manufacturing processes, a key method in developing 

CFC replacements. A comparison of industry benefits with 

the funding stream of NIST’s research program estimated 

a social rate of return of at least 433 %, and a benefit-tocost ratio of 4 to 1. This report was published in January 

1998, and the period studied was from 1987 to 1996. 

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

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

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

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

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

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

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

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

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

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

REFPROP is not explicitly specified in the standard, the 

ISO Working Group recognized the high quality and wide 

use of REFPROP and adopted the same models. A new 

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

replacing the “Refrigerant” of earlier versions indicates 

our direction: we will merge the NIST12 and NIST14 databases into REFPROP. Thus, a single database would 

encompass refrigerants, cryogenic fluids, hydrocarbons, and simple inorganics. 

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

and pointed their customers to 

REFPROP and CYCLE-D, and 

REFPROP has been licensed for 

use with the ASPEN process 

simulation package. 
12

Thermocouples: 

The NIST thermometry program includes both calibration services and research on thermocouples. Thermocouples are 

among the most commonly used sensors for monitoring and 

control of manufacturing processes. The annual sales of thermocouple products sold by the U.S. thermocouple industry 

(suppliers of wire and thermocouple assemblies) into the U.S. 

market are approximately $280 million. The incorporation of 

these devices into higher levels of product structures across a 

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

of $80 billion. 

Benefits were estimated based on surveys and interviews of 

the thermocouple industry and did not include the much larger, though diffuse, community of device users. Participants were asked to estimate the additional expenses that would have been 

incurred if NIST were to cease to provide primary calibration services. NIST expenditures included research on the fundamental and infrastructural aspects of thermocouple principles, 

measurement, and test methods as well as calibration services. This study, published in July 

1997, conservatively estimated the social rate of return to be 32 %, and a benefit-to-cost ratio of 

2.95. 

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. 

 

The next sections of this Report will delve into each of CSTL’s 14 programs individually, highlighting major activities and accomplishments in these areas for FY2002. Following the highlights of 

the 11 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. 

Gold versus platinum thermocouples are now readily 

available from several U.S. 

manufacturers. 

http://www.cstl.nist.gov/
13

Automotive and Aerospace 

Chemical and Allied Products 

Energy Systems

Environmental Technologies 

Food and Nutrition 

Forensics and 

Homeland Security 

Health and Medical 

Technologies 

Microelectronics 

Data and Informatics

Measurement Standards

B. CSTL Programs

Energy Systems

Biomaterials

Pharmaceuticals and 

Biomanufacturing 

Industrial and Analytical 

Instruments and Services 

Technologies for Future 

Measurements and Standards 
14
15

1. Automotive and Aerospace (Transportation) 

CSTL provides standards for 

emissions testing and flow 

measurements to help the 

transportation industry meet 

regulatory requirements. CSTL 

also works with the industry to 

provide physical and chemical 

property data that can be used 

in sophisticated computer models for next generation combustion engine design, and provides a 

new generation of chemical standards and reference data to support advanced materials such as 

super alloys for high technology jet engines. 

SRMs for Low Level Automotive Emissions 

W.J. Thorn III (839) 

Stakeholders in the American Industry/Government Emissions Research (AIGER) group are 

working together to facilitate the automobile industry meeting more stringent 2003 Federal Tier II 

and California LEV II emission regulations. New more 

efficient catalysts and computer optimized 

combustion parameters have reduced tailpipe 

pollutants to very low μg/g (ppm) levels. These 

emission levels are further diluted by air into constant 

volume sampling bags or through the use of new 

mini-diluter technology while performing the Federal 

Highway Test on a chassis dynamometer. 

Development of lower concentration nitric oxide 

SRMs has been given the highest priority by AIGER 

members and specialty gas suppliers. 

In 1998, NIST anticipated the AIGER need for 

significantly lower nitric oxide standards by 

partnering with Scott Specialty Gases to solve the 

instability of the lowest existing SRMs (5 μg/g and 

10 μg/g) and to produce new stable nitric oxide 

standards between 0.5 μg/g and 1.25 μg/g. These 

new infrastructure standards have exhibited 

excellent stability for more than 3 years. In 2001, 

AIGER members including the U.S. EPA, California 

Air Resources Board (CARB), General Motors, 

Ford, and Daimler-Chrysler requested that NIST 

develop two new nitric oxide SRMs at 0.5 μg/g and 

1.0 μg/g. With the assistance of AIGER member funding, NIST and Scott Specialty Gases 

collaborated to gravimetrically blend 40 cylinders of each 

new nitric oxide concentration, 80 cylinders total. NIST then 

performed a 100% analysis on the 80 mixtures to verify that 

each cylinderís nitric oxide concentration is within ± 1 % 

relative. Fifty-four of these mixtures have now been given to 

NIST to be certified over the next two years as new SRMs, 

while the remaining twenty-six mixtures were distributed to 

AIGER members who ordered them, to serve as interim 

standards until the new SRMS are available in December, 

NIST and the Motor Vehicles 

Manufacturers have worked 

together since 1975 to develop 

sixty gaseous Standard Reference 

Materials (SRMs) against which all 

EPA mandated fuel economy and 

mobile source emission 

measurements must be traceable, 

by federal law. 

This work has resolved the 

technical issues related to the 

stability of low-level NO, and 

provided AIGER members with 

interim standards. NIST will 

also provide SRMs in the near 

future to help industry meet 

lower emission regulations.

In 2004, California’s zeroemission vehicle law will take 

effect, calling for 10% of an 

automaker’s vehicles sold in 

that state to be zero-emissions 

vehicles. 

Automobile 

Exhaust/Emissions 

System 
16

2003. Discussions are ongoing with AIGER to upgrade the NTRM protocol to allow NTRM gas 

standards to replace NIST SRMs for non-reactive emission measurements. This could free NIST 

resources to work on standards of other AIGER priority pollutants such as 0.1 μg/g methane. 

Critically Evaluated Fourier-Transform Microwave Spectroscopy for Real-Time 

Measurements of Automotive Exhaust Emissions 

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

The high spectral resolution and high sensitivity of Fourier-transform microwave (FTMW) 

spectroscopy suggest that the technique can provide real-time response and unambiguous 

identification of vapor phase analytes with permanent dipole moments. The principal goal of the 

present program is to critically evaluate the 

techniqueís potential as a reliable and robust 

tool for quantitative measurements of trace 

gases. Current work has focused on 

characterizing and improving the signal 

intensity stability and optimizing the 

instrument for gas samples with air as the 

primary balance gas. This project was 

originally initiated to address the automobile 

industryís need to identify and quantify 

oxygenates at trace levels in auto exhaust 

emissions. 

 

FY 2002 work has focused on characterizing the 

instrument response over extended periods and 

optimizing the system performance. Initial evaluation 

revealed that measured peak positions are 

reproducible permitting definitive species identification. 

The signal intensities, however, varied substantially 

over long measurement periods (12 h), preventing 

reliable quantitative measurements. After several 

modifications, the signal intensity drift has been 

reduced to ≤10 % over 48 h. The maintenance of the 

instrument had been significantly reduced as well. 

Additional modifications in the sample delivery system 

have further improved the systemís sensitivity for 

samples prepared in nitrogen-based matrices 

compared to previous work, which required rare gas 

based matrices. As work to optimize the instrument 

performance continues, an effort will also focus on evaluating 

the capability of FTMW spectroscopy to measure analytes in 

gas matrices that more closely match the specific applications, 

such as systems with high concentrations of carbon dioxide and 

water. 

Fourier-transform microwave (FTMW) 

spectroscopy is being investigated as a 

quantitative analytical technique to meet 

the need for an improved analytical tool 

that provides real-time measurements of 

trace-level vapors. These tools are critical 

for addressing pressing industrial issues 

such as global climate change and the 

detection of hazardous emissions. 

Cutaway view of FTMW 

spectrometer 

Success of this work will 

also impact many other 

critical applications 

including the detection of 

chemical warfare agents. 
17

SRMs for Hydrogen in Titanium 

R.M. Lindstrom, R.L. Paul, and H.H. Chen-Mayer (839) 

Hydrogen embrittlement can be a serious problem for many metals, leading to massive structural 

failures in extreme cases. Therefore the control of hydrogen in the manufacturing process is 

crucial. Hot-extraction measurement methods used in industry need to be calibrated with 

accurate working standard materials. 

Industrial analytical methods for hydrogen employ hot extraction in vacuum or inert 

gas, followed by detection of the hydrogen by gas chromatography or mass 

spectrometry, for example the ASTM Standard Test Method E1447 for hydrogen in 

Ti alloys. These methods in practice require independently produced, matrixmatched metal standards for calibration, and reference materials for continued 

validation of the analytical procedure. 

To respond to this need, NIST began the 

development of a Hydrogen in Titanium SRM. In 

the SRM preparation, titanium alloys are loaded 

with a known quantity of hydrogen by carrying the 

reaction out in a closed system with known 

volume. The first Standard Reference Material 

SRM 2453, Hydrogen in Titanium (100 mg of 

hydrogen per kg of titanium) has been prepared by 

this method. The amount of hydrogen added to the 

metal was measured by the volume of gas 

absorbed and also by the mass gain. Promptgamma activation analysis (PGAA) and neutron incoherent 

scattering (NIS) were used to measure hydrogen in the metal 

before and after loading. Two additional bracketing materials are 

required to check calibration linearity and provide check samples 

at levels significantly above and below the critical level of ≈ 100 

mg/kg. SRMs 2452 and 2454 are in preparation at target mass 

fractions of 50 mg/kg and 200 mg/kg, respectively. 

A combination of wellcontrolled preparation 

procedure and neutron 

based characterization 

gives assurance that these 

SRMs are traceable to 

fundamental parameters. 

The aerospace and, increasingly, the 

automotive and consumer-goods 

industries employ titanium alloys 

because of their excellent 

combination of high strength, light 

weight, and high temperature 

properties. NIST SRMs help ensure 

that fabricated components in fact 

have these desired properties. 
18
19

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. 

Biomarkers of Oxidative DNA Damage Used to Detect Genetic Changes in Tissue 

Engineered Skin 

H. Rodriguez, P.E. Barker, C.D. O’Connell, and M.M. Dizdar (831); P. Jaruga (UMBC, MD), 

and M. Birincioglu (Inönü University, Turkey)

Fundamental to many tissue-engineered devices are issues of inflammation associated with how 

biological cells respond to a given matrix or when inserted into the body. It has become 

increasingly clear in recent years that oxidative damage to 

DNA plays an important role in a number of disease 

processes. Liquid chromatography/tandem mass 

spectrometry (LC/MS/MS) and liquid chromatography/mass 

spectrometry (LC/MS) have emerged as new techniques for 

the measurement of modified nucleosides in DNA. Using gas 

chromatography/mass spectrometry (GC/MS), a total of five 

genomic DNA biomarkers in tissue-engineered skin 

(TestSkin II®, Organogenesis, Inc) was screened and the 

levels of damage compared to control cells, including 

neonatal human dermal fibroblasts, neonatal human 

epidermal keratinocytes, cultured HeLa cells, and 

commercially available calf thymus DNA. The biomarkers consisted 

of FapyAdenine, FapyGuanine, 8-OH-Guanine, 5-OH-Uracil, and 5-

OH-Cytosine. For 8-OH-Guanine (a free base), its nucleoside form 

(8-OH-dGuanosine) was also monitored using LC/MS. 

Results showed that the level of oxidative DNA damage was found 

to be at background/endogenous levels (approximately 1 to 10 

modified molecules/106

 DNA bases). Nearly identical results were 

obtained when measuring the nucleosides with LC/MS. Accuracy of 

the measurements was achieved using stable isotope-labeled 

analogues of modified 

DNA bases as internal 

standards. The results 

show that the obtained 

tissue-engineered skin did not contain any elevated 

levels of oxidative DNA damage. In order to assure 

that such composite materials are free of genetic 

changes that might occur from oxidative stress during 

the manufacturing, storage, or transportation of the 

product, we will consider other cellular biomarkers 

Scientific studies have 

shown that free radicals are 

culprits causing many 

diseases of the older 

population. The most 

common biomarker for 

assessing free radicalinduced oxidative stress in 

living cells is oxidative 

damage to DNA. 

Oxidative damage to DNA has been 

implicated in cancer and several 

neurodegenerative diseases such as 

Amyotrophic Lateral Sclerosis (Lou 

Gehrig's disease), Alzheimer’s disease 

and Parkinson’s disease. Furthermore, 

the accumulation of oxidative damage 

to DNA especially in non-dividing cells 

has been postulated to be responsible 

for the degenerative effects of aging. 

The total market for the 

regeneration and repair 

of tissues and organs is 

estimated to be $25 

billion worldwide. 
20

that could be used during the development phase of tissue-engineered materials to ensure that 

cells have not undergone any inflammatory response during the development or shipment of the 

product.

Thin Films of Collagen Effect Smooth Muscle Cell Morphology 

A.L. Plant, J.T. Elliott, and J.T. Woodward, (831); A. Tona (Brown Univ.), and P. Jones 

(Univ. of Colorado Health Sciences Center) 

Collagen is the most abundant extracellular matrix protein in 

the body, and it is an essential component of many tissueengineered devices. It can assume different molecular and 

supramolecular structures, depending on the conditions under 

which it is prepared for cell 

culture. Standardized protocols 

for collagen use in cell culture 

studies do not currently exist, 

and rarely are the final matrices 

characterized. Furthermore, the 

cellular response to different 

forms of collagen is poorly 

studied. 

CSTL researchers have 

developed a reproducible 

method for applying collagen to surfaces on which cells can be 

grown, and to characterize the resulting thin films of collagen 

protein with respect to molecular structure and cellular 

response. Using automated quantitative microscopic analysis 

we showed that the morphology and proliferation of vascular 

smooth muscle cells is determined by the intermolecular 

interactions of collagen in these thin films. 

Biophysics from First Principles 

A.M. Chaka (838) and F. Seno (Univ. of Padova, Italy)

An international, multidisciplinary conference on 

Biophysics from First Principles was organized to 

bring together theoretical and experimental 

researchers from physics, chemistry, biological 

sciences, and industry to foster interdisciplinary 

approaches to the study of living systems. 

These researchers have 

shown that thin films of 

collagen can be 

reproducibly formed 

under conditions that 

produce either fibrillar 

or monomeric collagen, 

which mimic the healthy 

and diseased artery, 

respectively.

Next Steps: 

Other rugged and reproducible thin films will be 

developed as essential reference surfaces on 

which smooth muscle and other cells can be 

examined for analysis of their intracellular 

signaling in response to their culture 

environment. 

The size of cells on thin 

films (solid lines) was 

compared to cells on 

thick gels (dashed lines) 

using automated 

quantitative microscopy 

Conference on Biophysics from First 

Principles: From the Electronic to the 

Mesoscale, September 7-12, 2002, San 

Feliu, Spain
21

CSTL researcher Anne Chaka was elected 2002 Program Chair for 

Psi_k, the European theoretical physics organization. She also served 

and will continue to serve on the steering committee for the new 

EURESCO Computational Biophysics: Integrating Theoretical Physics 

and Biology Conference series. 

The principal focus was on the application of theoretical physics – 

from first principles to classical simulations, as well as statistical 

approaches – to understanding biological processes. Challenges 

facing industry and the environment were presented by invited plenary 

speakers from DuPont (Daniel Kleier, pesticide invention), BASF 

(Hugo Kubinyi, drug design and virtual screening), and the U.S. 

Environmental Protection Agency (EPA) (James Rabinowitz, 

understanding health effects of environmental chemicals). The 

maximum capacity of 135 researchers from academic and government institutions around the 

world attended, as well as industrial participants from companies such as DuPont, BASF, 

Unilever, Toshiba, and IBM. There was a waiting list of over 150 people hoping to attend. The 

conference was deemed so successful and the 

interdisciplinary need so timely that the European 

Science Foundation (ESF) has decided to initiate 

a new conference series entitled “Computational 

Biophysics: Integrating Theoretical Physics and 

Biology” to be held every two years. 

Physicists and biologists have few opportunities 

to interact, yet such interaction has the potential to contribute greatly to progress in both fields. 

No single simulation or experimental approach will provide all the answers, and solving life 

science problems requires the skills and knowledge from across traditional disciplines. Advances 

in physics, such as electronic structure and ab initio dynamics, have the potential to play a vital 

central role in the study of biological systems. The 

size and complexity of biological problems have 

driven development of classical approaches such as 

molecular mechanics and force field methods, and 

the calculation of free energy of solvation. 

Theoretical models and numerical simulations from 

both disciplines need to be integrated to achieve the 

synergy that is necessary to make real predictions. 

This conference represents the first time leading 

researchers in the field of biological simulations (M. 

Karplus, K. Schulten, M. Klein), experimental biology (C. Dobson, H. Schrempf), theoretical 

physics (M. Parrinello, R. Car, B. Lundqvist), and experimental physics (B. Kasemo, W. Zinth) 

have been brought together for a week-long, intensive conference and discussions. Several 

challenges were identified, such as a continuing mechanism to foster interdisciplinary research 

between biologists and physicists. The importance of chemistry as a key link between biology 

and physics was also stressed. The major challenge identified is the integration of time and 

length scales, particularly to treat complex problems such as protein folding, biochemical 

pathways, toxicology, and ion transport through membranes. 

CSTL’s Computational Chemistry 

Group will develop plans to address 

key modeling challenges identified 

such as integrating time and length 

scales, validating current simulation 

methods, mesoscale phenomena, and 

improving robustness of biological 

simulations through increased use of 

first principles techniques. 

Session topics included: Understanding 

Biological Mechanisms; Transition Metal 

Chemistry; Predicting the Biological 

Activity of Chemicals; Structure and 

Function of Biological Macromolecules; 

and Molecular Interactions and Solvation: 

Short- and Long-Range Forces. 
22
23

The determination of 

enantiomeric composition is 

a critical quality issue in the 

pharmaceutical industry. 

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, CSTLís 

fundamental work in enzyme characterization promotes the transition to 

biomanufacturing leading to more environmentally sustainable 

manufacturing

Enantioselectivity of Three Sulfated Cyclodextrins for the Capillary Electrophoretic 

Analysis of Pharmaceutical Products 

K.W. Phinney (839) 

Capillary electrophoresis (CE) is increasingly being used for the determination of enantiomeric 

composition of chiral drugs. Cyclodextrins (CDs) and their derivatives remain the most widely 

used chiral additives in CE, and many derivatized cyclodextrins are now commercially available. 

Several types of anionic CDs have been developed, including a number of CDs bearing sulfate 

groups. Differing modes of preparation lead to materials 

that vary in both composition and enantioselectivity. 

Although this range of enantioselectivity provides some 

flexibility in method development, it can also complicate 

the choice of an appropriate chiral additive for the desired 

separation. 

Three commercially available sulfated cyclodextrins having similar numbers of sulfate 

substituents were selected for evaluation (see figure). The mixed isomer sulfated CD proved to 

be applicable to the greatest number of the target analytes. Hence, the use of this chiral additive 

could increase the likelihood of successful method development. On the other hand, the uniform 

composition of the single isomer sulfated CD could be beneficial for ensuring method 

reproducibility. The lowest success rate for the target analytes was obtained with the 

sulfobutylether CD, but a significant degree of enantioresolution was achieved for some of the 

analytes. The number, type, and position of the anionic substituents clearly play a role in the 

enantioselectivity of these CDs. 

 

The length of time required to 

develop analytical methods 

has remained a bottleneck in 

the drug development 

process. Detailed studies of 

enantioselectivity such as this 

one provide a knowledge base 

that simplifies chiral method 

development. 

Structures of sulfated cyclodextrins

Increased demand for 

new products has 

replaced price as the 

primary force in the U.S. 

pharmaceutical 

industry’s overall 

growth. 
24

Development of an NMR-Based Drug Screening Approach for GPCRs 

 K. D. Ridge and J. P. Marino (831/CARB); N. G. Abdulaev (CARB/UMBI); and D. M. 

Brabazon (Loyola College, MD) 

Although a crystal structure for the integral membrane G-protein coupled receptor (GPCR) 

rhodopsin is now available, portions of the cytoplasmic surface are not well resolved and the 

structural basis for its interaction with the G-protein (Gt) is unknown. The lack of high-resolution 

structural data for this pharmacologically tractable class of 

integral membrane protein receptors remains a major gap in 

our knowledge. Previous studies using soluble mimics of 

light-activated rhodopsin have shown that grafting defined 

segments from the cytoplasmic region onto a surface loop of 

thioredoxin is sufficient to confer Gt activation. To assess 

whether these mimics can also provide structural insights 

into the interaction between light-activated rhodopsin and Gt, 

the ability of a thioredoxin fusion protein comprised of 

rhodopsinís second and third cytoplasmic loops to bind Gtα -

subunit (Gtα) peptides was examined by NMR spectroscopy. Titration experiments show that 

peptides corresponding to the carboxyl-terminus of Gtα bind to this soluble mimic and undergo 

small but significant structural changes in the bound 

state. Remarkably, the peptides adopt a C-cap like 

structure similar to that observed upon the binding of Gtα

peptides to intact, light-activated rhodopsin. These 

findings suggest that this functional mimic of lightactivated rhodopsin is also a structural mimic for the 

signaling state and provides a novel solution-based 

structural assay for screening small molecule inhibitors 

that potentially disrupt activated GPCR/G-protein 

interactions. 

The Protein Data Bank 

T.N. Bhat and G.L. Gilliland (831/CARB); and P. Fagan (TS) 

The Protein Data Bank is the single worldwide repository for the processing and distribution of 3-

D biological macromolecular structure data. Many secondary sources of information are derived 

from PDB data. Structural biologists, medicinal chemists, and other biological and biomedical 

scientists from industry, academia, and private research efforts that engage in protein 

engineering, structural genomics, rational drug design, 

protein stability, and other studies of biological 

macromolecules require information on the structures of 

biological macromolecules. These studies, which use 

the three-dimensional structural data, lead to new drugs, 

new proteins with desired properties for commercial 

applications, or new insight into biological processes. 

The coordinates are also essential for bioinformatics 

efforts since it is well recognized that the threedimensional structure of a protein is more generally 

conserved than is the amino acid sequence. The goals 

of the PDB, the systems in 

place for data deposition and 

access, how to obtain further 

Comprehensive 

understanding of the 

molecular details governing 

activated GPCR/G-protein 

interactions is important to 

the development of drugs 

that alleviate a number of 

human diseases. 

The approaches developed here 

offer a promising strategy for 

“solubilizing” the functions of other 

GPCRs in order to expedite a 

structural understanding of Gprotein binding and specificity and 

to the application of drug discovery 

efforts focused on this interaction. 

The PDB provides industry and 

the research community with 

three-dimensional structural data 

of biological macromolecules that 

can be used for basic research 

and for countless applications in 

the pharmaceutical and 

biotechnology industries. 

http://www.pdb.org/
25

information, and plans for the future development of the resource 

are described in a special issue of Acta Crystallographica D

dedicated to crystallographic databases. The article will also be 

published as a chapter in a forthcoming book ìStructural 

Bioinformaticsî to be published by John Wiley & Sons (Philip E. 

Bourne and Helge Weissig, Editors). 

Rapid Identification of Compounds That Inhibit Retroviral RNA-Protein Complexes Using 

Fluorescence and NMR Spectroscopy 

E.S. DeJong and J.P. Marino (831/CARB); C. Chang and M.K. Gilson (CARB/UMBI) 

RNA and RNA-protein complexes can provide potentially powerful targets for the regulation of 

gene expression and inhibition of viral/bacterial infection. For instance, the inhibition of proteins 

that are involved in mRNA processing, transcription, or retroviral replication could provide 

powerful new drug targets to combat viral infections, fight 

cancer, or enhance the effectiveness of existing 

chemotherapeutic agents. To drive RNA-based drug 

discovery, general approaches are required for detecting 

and quantifying nucleic acid-protein interactions that can 

be used as high-throughput screens (HTS) and for 

obtaining rapid structural information to guide rational 

drug design. Using a 2-AP fluorescence perturbation 

screen, we have identified small heterocyclic aromatic 

compounds that interact 

with high affinity (low μM 

to nM range) with the Rev Responsive Element (RRE) of HIV-1, a 

potential RNA target for antiviral drugs. A few of the compounds 

identified through the 

fluorescence screen have been 

further shown to effectively 

compete with an arginine-rich 

peptide, derived from Rev, for 

binding to RRE (KI in the μM to 

nM range). NMR structural analysis of one of the identified 

inhibitors, proflavine, bound to RRE has revealed a 2:1 

(proflavine:RRE) binding stiochiometry, with the two proflavines 

stacked one on top of the other in the binding site. NMR 

experiments also demonstrate that binding of proflavine 

stabilizes the purine-rich high-affinity Rev binding site of RRE 

in a manner analogous to the native Rev-RRE interaction. 

 

Efforts of the NIST staff have led to improved 

capabilities for querying the data, thereby enabling 

fuller utilization of the data and the resource. 

The development of specific 

inhibitors of protein-RNA 

complexes is of significant 

interest to the pharmaceutical 

industry because these 

complexes provide potentially 

powerful targets to regulate 

gene expression and inhibit 

bacterial/viral infection. 

Future Plans: Through collaboration with researchers at the National Cancer Institute,

we plan to apply our methods to screen large public domain libraries of compounds 

in a search for new lead compounds against retroviral RNA targets, which will then 

be optimized using rational design strategies based on NMR structural analysis.

NIST researchers have 

identified a new class of 

compounds that target the 

HIV-1 retroviral RRE-Rev 

RNA-protein interaction, 

providing new lead 

compounds in the search 

for antiviral therapeutics.

HIV 1 Cross Section

Molecule of the Month 

This structure shows the 

filter portion of a bacterial 

potassium channel. 

PDB, Feb 2003 
26

Structural and Biochemical Studies of Enzymes Along the Chorismate Pathway 

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

Industrial production of drugs, new biopolymers, and indigo dyes, can be facilitated by increased 

understanding of the aromatic amino acid synthesis pathway. The increased ability to manipulate 

this pathway through metabolic and protein engineering will save non-renewable, petroleumbased feedstock chemicals. This study provides a 

description of the enzymes involved in the chorismate 

metabolic pathway by solving their three-dimensional 

structures, modeling the mechanisms of the chemical 

transformations, and mapping pathway control nodes 

involved in the biocatalytic conversion of glucose to 

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

precise enzymatic mechanisms and relationships between 

the structure and the physical properties be predicted. Most 

recently, the structure of the product of gene phzD from Pseudomonas aeruginosa has been 

solved. The phenazines are biologically active (antibacterial, antfungal, antitumor) aromatic 

products synthesized mainly by Pseudomonas and Streptomyces as part of their chorismate 

pathways. The phzD gene product is involved in the pathway in Pseudomonas in the production 

of phenazine-1-6-dicarboxylic acid. 

Target Site of Intron Gain Inferred by a System for Phyloinformatic Analysis (SPAN) 

A.B. Stoltzfus (831/CARB); W-G. Qiu (CARB/UMBI), and N. Schisler (Pomona College), 

Due to its proven utility, comparative analysis of sequences (comparing homologous genes, 

proteins, or genomes to derive clues about structure and function) plays a central role in many 

areas of biotechnology (genome annotation, drug target discovery, biomolecule engineering, 

medical genetics). The most rigorous methods of comparative 

analysis are evolutionary ones, based on identification and 

alignment of homologous sequences, inference of a 

phylogenetic tree, and phylogenetic reconstruction of changes 

associated with biologically relevant differences. Due to its 

complexity and its reliance on human supervision, this type of 

analysis conventionally is done by an expert, one sequence 

family at a time. Our goal is to develop a software system that will allow evolutionary analyses of 

sequence features to be carried out using hundreds of sequence families at a time. Such a 

System for Phyloinformatic Analysis (SPAN) will provide the bioinformatics community with a 

rigorous high-throughput method of comparative sequence analysis. The test-bed for 

development of SPAN is a set of problems regarding the evolution of introns that have withstood 

a decade of analysis using more conventional methods. On the hypothesis that introns are 

gained non-randomly, we aim to infer a precise intron ìtarget siteî, and then use this to predict 

various aspects of the distribution of introns. In place of the expert judgement that would be used 

conventionally to tailor an evolutionary analysis for each sequence family, we use automated 

methods that assess the reliability of information, so as to allow an objective ìhigh-gradingî 

approach in which conclusions are based on only the most reliable parts of the analysis. Based 

The elucidation of pathways 

to aromatic biologically 

active molecules can provide 

pharmaceutical researchers 

with a better understanding 

for the amplification or 

inhibition of the production 

of these molecules. 

Comparative analysis of 

sequences plays a 

central role in many 

areas of biotechnology 

The structure of the 

product of gene phzD 

has been solved, and the 

active site of the 

molecule has been 

identified.
27

on this kind of analysis applied to a set of 10 sequence families, we find clear evidence that 

introns are gained non-randomly at sequences favoring the nucleotide CAG^GT. 

Data from ten sequence families comprising 677 

genes were collated, the sequences were 

multiply aligned, and family trees were inferred. 

The resulting data sets were loaded into the 

SPAN database and augmented with 

probabilities of ancestral intron states estimated 

using a Bayesian method developed at CARB. 

These procedures are not fully automated as 

yet. Database tools were developed to identify 

patterns of intron gain. In place of the expert 

judgement that might normally be used in this 

kind of analysis to tweak alignments and trees, and to ignore dubious data points, our system 

allows us to implement an objective ìhigh-gradingî 

approach based on explicit measures of the reliability of 

alignment columns, phylogenetic tree branches, and 

reconstructed events of intron gain and loss. The resulting 

intron ìtargetî pattern shown here is based on the most 

trustworthy parts of a large set of data. Preliminary results 

suggest that this pattern can account quantitatively for 

patterns in the distribution of intron phases that previously 

were poorly understood. 

 

Theoretical Studies of Enzyme Mechanisms 

M. Krauss (831) 

Class B lactamases are a prime defense mechanism of pathogens against lactam antibiotics and 

are of great interest medically. Knowledge of the structural and energetic details of the reaction 

path on a molecular level would be useful in the design of inhibitors. The reaction mechanisms of 

two enzymes, zinc lactamase and chorismate mutase, that 

are representative of their class were studied theoretically. 

The reaction mechanism of the di-zinc lactamase enzyme 

has not been determined before at the molecular level either 

experimentally or theoretically. An ab initio quantum 

mechanical and molecular 

mechanics (QMMM) theoretical 

method for calculating reaction 

paths in an enzyme has been 

applied to chorismate mutase in 

collaboration with scientists at the 

NIH. This enzyme has been the focus of many theoretical studies but 

the questions on details of the structure 

and energetics along the reaction path 

have persisted. 

Zinc lactamase possesses a flexible flap 

that forms part of the active site in 

binding of the substrate. This flap has to move approximately 10 Å 

and the theoretical solution requires a combination of standard 

molecular dynamics codes and chemical intuition. The reaction path 

for the hydrolysis of the antibiotic lactam is calculated to involve 

highly polarized waters connected in hydrogen bonded networks in 

This system will be useful in 

deciphering genome organization, 

since it will lead to a much greater 

understanding of one of the 

central features of eukaryotic 

genome organization, namely the 

splitting of genes into introns and 

exons. 

Global optimization and then 

ab initio dynamics on the 

fundamentals of enzyme 

reactivity are required to 

compare the same enzymes 

from different organisms to 

ultimately understand 

specificity of a specific 

enzyme in a given organism.

Zinc Lactamase

Chorismate Mutase 
28

the highly ionic active site. A range of reactive intermediates is described for the reaction. It is 

shown that the intermediates can form by proton transfers if the initial hydrolysis products are not 

removed from the active site of the enzyme by rapid solvation. 

The ab initio QMMM theoretical method for calculating reaction paths in an enzyme allows for the 

optimization of the entire enzyme providing the means for 

accurate calculation of the reaction path and also new insight into 

the interactions between the active site and the protein 

environment. One new insight is the observation that the protein 

environment around the enzyme active site does not change 

appreciably during the reaction. In another aspect of this study we 

have calculated the kinetic isotope effects of an enzyme reaction 

in the active site of the enzyme for the first time. 

 

CSTL scientists have used 

theoretical studies to gain 

insight into the reaction 

mechanisms of medically 

important enzymes.
29

4. Chemical and Allied Products 

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. 

Critical Benchmark Standards for the Cement Industry 

J.R. Sieber and A.F. Marlow (839); B.S. MacDonald and C.N. Fales (TS); S.D. Leigh and J.H. 

Yen (ITL); D. Broton (Construction Technology Laboratories); and S. A. Wilson (USGS) 

For several years, CSTL researchers have worked with Construction Technology Laboratories 

and the United States Geological Survey on SRM projects to benefit the cement industry. These 

materials represent cement compositions produced around the world with materials obtained in 

North America, Europe, and the South Pacific. 

Variety of composition is important because there 

are many cement products for a myriad of uses. In 

addition, product quality labs use SRMs with 

instrumental methods like X-ray fluorescence 

(XRF) and atomic absorption as well as classical 

chemical methods of analysis. Portland cement 

SRMs are used worldwide 

because they are required by 

ASTM International in ASTM C 

114-99 Standard Test Methods for Hydraulic Cement. ASTM C 114 requires at 

least six SRMs; therefore, it was necessary to replace the SRM 1880 series. 

The final four portland cement SRM renewals were completed this year: SRM 1881a Portland 

Cement blended with slag and fly ash, SRM 1886a White 

Portland Cement with Low Iron, SRM 1888a Portland 

Cement, and SRM 1889a Portland Cement Blended with 

Limestone. All renewal SRMs are certified for more 

constituents and properties than the original issues. The 

work was described in a paper accepted for publication in 

Cement and Concrete Research: ìStandard Reference 

Materials for Cementsî, J. Sieber, D. Broton, C. Fales, S. 

Leigh, B. MacDonald, A. Marlow, S. Nettles, and J. Yen. 

NIST, ASTM International, and the 

cement industry have long been 

partners in the creation of SRMs and 

research into the characteristics of 

cement and concrete products. SRMs 

underpin product quality control for 

the cement industry worldwide and 

have done so for over 50 years. 

Due to strong reliance of the 

industry on XRF for elemental 

analysis, the members of this 

project team convinced ASTM 

International to form a task 

group to create a new method 

specifically for XRF analysis of 

hydraulic cement. 

A modern cement plant in 

Chanute, Kansas. 

Often referred to as the keystone 

industry because nearly every 

sector of the manufacturing 

economy uses its products, the 

U.S. chemical industry ranks 

among the worldís leading 

industries, accounting for an 

estimated one-quarter of total 

world chemical production. 
30

Critical Benchmark Standards for the Metals Industry 

J.R. Sieber, A.F. Marlow, J.D. Fassett, G.C. Turk, R.M. Lindstrom, M.L. Salit, and M.R. 

Winchester (839); and J.R. Rumble (TS) 

The metals industry is implementing a system for product quality control based on ISO/IEC 

17025:1999, General requirements for the competence of testing and calibration laboratories. 

Product control laboratories gain accreditation by 

demonstrating traceability to national reference 

materials using international standards. 

Producers must also comply with performance 

benchmarks imposed by customers. They 

demonstrate comparability to customer labs and 

other producer labs by participating in proficiency 

test programs. Producers and customers are 

looking to creators of standards and certified 

reference materials to provide the infrastructure 

with which to establish the proficiency of all 

parties. Industry labs need help with certified 

reference materials, better analytical methods, 

and uncertainty estimates. 

Five SRMs for metals, alloys, and related materials were completed in FY 02. These SRMs 

benefit the copper, steel, and aluminum segments of the industry and their customers in 

aerospace, automotive, electrical, semiconductor, and construction industries. 

The level of interaction between NIST and industry will be maintained with emphasis on solving 

the problem of high demand for an ever-growing variety of new certified reference materials 

versus high development costs, long lead times, and low sales prospects for any single reference 

material. NIST scientists will continue to develop matrix-independent methods and work to 

transfer the technology to the metals industry. 

The development of benchmark 

standards requires in-depth 

knowledge of the industry and close 

cooperation with industry 

representatives through ASTM 

International, commercial reference 

materials producers, and corporate 

laboratories. NIST gains industry 

input on prioritization of SRM projects 

and helps commercial producers 

meet the increasing demand for 

reference materials. 

Accomplishments 

• SRMs C1251a, C1252a, C1253a, Phosphorus Deoxidized Copper (completed) 

• SRM 699 Alumina (Reduction Grade) (revision of SiO2 value, completed) 

• SRM 133b Cr-Mo Steel (renewal) 

• Representation in ASTM Committee E01 on Analytical Chemistry of Metals, Ores and 

Related Materials for prioritization of SRM requests and method development. 

• Assisted the Interlaboratory Analysis Program of Analytical Reference Materials 

International by providing training and guidance for CRM production. 

• Established traceability of X-ray fluorescence and atomic emission spectrometry to the 

mole. 

Electron Beam Cold Hearth Refining 

of specialty metals. In this case, Ti 

bars are created in a continuous 

refining process. 
31

Cylinder Filling Ratios for Compressed Liquids 

M.L. Huber and D.G. Friend (838) 

 

As the UN Committee of Experts on the Transport of Dangerous 

Goods performs its mandated study on safety issues related to 

international commerce, the Department of Transportation (DOT) 

provides input related to domestic industrial requirements and the 

domestic regulatory environment. NIST has been asked to assist 

in the development of cylinder filling ratio guidelines to be used in 

domestic and international commerce of certain fluids. 

 

The filling ratio definition we used in this work is the 

ratio of the mass of a fluid to the mass of water at 15 

 ̊C that would completely fill a pressure receptacle (this 

definition is in the draft of UN Model Regulation on the 

transport of hazardous materials). Depending on the 

fluid involved, there are different restrictions placed on 

the filling ratio to ensure safety. For example, it is 

important that cylinders exposed to high temperatures 

that may be encountered during storage or 

transportation do not develop high pressures that could 

exceed safety limits. Therefore, it is important to be 

able to determine the filling ratio accurately for a wide 

variety of fluids. For this project, models were developed to compute the filling ratio using several 

different methods. When available, high-accuracy equations of state, such as those in NIST 

Standard Reference Databases, were used. For fluids where dedicated, high-accuracy equations 

of state were not available, a predictive model based on the theory of extended corresponding 

states was used. This method has been used successfully in the past for modeling 

hydrocarbons, cryogens, and refrigerants. The 

results were verified by comparison with 

experimental data where available and existing or 

proposed international or domestic guidelines. 

This project provides a reliable model for the 

calculation of the filling ratio for liquefied gases 

required for the safe handling of gas cylinders. The 

results are expected to be incorporated into a UN 

Model Regulation and to be used in international and domestic commerce of certain hazardous 

materials. 

 

Double-Sinker Densimeter for High-Accuracy Fluid Density Measurements 

M.O. McLinden (838) and C. Lˆsch (Rubotherm Pr‰zisionsmesstechnik, Germany) 

Density as a function of temperature and pressure is one of the most important properties for the 

determination of a thermodynamic equation of state of a fluid. Density is also vital in fluid 

metering applications in research and industry. A new apparatus for high-accuracy fluid density 

measurements over wide ranges of temperature, pressure, and density has been completed and 

put into operation (ñ180  ̊C to +250  ̊C; 0 MPa to 40 MPa; 5 kg/m3 to 1700 kg/m3

). This new 

apparatus extends our capabilities to higher temperatures and lower densities and reduces the 

uncertainties by an order of magnitude. These capabilities will be used to provide data on key 

industrial fluids and mixtures and develop Standard Reference Materials (SRMs) for fluid density. 

CSTL researchers provided dependable 

values of the filling ratio for a wide 

variety of industrially important fluids 

such as refrigerants, hydrocarbon 

gases, welding gases, semiconductor 

processing fluids, odorants, and other 

commercially available fluids. 
32

This new instrument (pictured) employs an Archimedes 

(buoyancy) technique using two sinkers immersed in the 

fluid of interest. Sinkers and magnetic suspension 

coupling comprise the heart of the densimeter. The 

electromagnet at the top hangs from the balance and 

levitates the permanent magnet below it. The use of two 

sinkers leads to a cancellation of effects, such as 

surface adsorption, which 

can affect the accuracy of 

single-sinker techniques, 

making this instrument 

especially useful for the measurement of complex mixtures and 

vapors near saturation. The density measuring system was 

developed in collaboration with Rubotherm, Inc. of Bochum, 

Germany. Their components were integrated with the bulk of the 

system, which was fabricated in the NIST Instrument Shops. The 

magnetic suspension coupling has proven stable and reproducible to 

better than 5 μg; yielding a reproducibility in density of 0.0005 kg/m3

(i.e., 5 ppm or less for gases of moderate density as well as liquids). 

The volumes of the sinkers have been determined to an uncertainty 

of 20 ppm by comparison to silicon density standards. The final 

uncertainty in the density depends on both the weighings and the 

sinker volumes, and we are confident that we will achieve our goal of 

0.01 % (100 ppm) uncertainty in density. Although originally 

designed for measurements on low-density gases, the apparatus has 

proven capable on liquids with densities as high as 1700 kg/m3

. 

Environmental Control of α-Cr2O3 (0001) Surface Chemistry 

A.M. Chaka (838), N. Harrison (Imperial College, U.K.), A. Wander (Daresbury Laboratory, 

U.K.), and X.-G. Wang (GM Research Laboratories) 

 

Chromium oxide is very important for a wide variety of technological applications such as 

catalysis and surface passivation of metal alloys like stainless steel. Chromium is also added to 

aluminum alloys because it will segregate to the surface, 

oxidize, and provide an almost universal foundation for 

primer and coatings for subsequent surface engineering. 

Despite its importance, the surface structure and 

stoichiometry of chromium oxide is not well understood 

because its insulating character renders analytical 

techniques based on electron spectroscopy problematic. It 

is also difficult to obtain clean, well-ordered surfaces for 

investigation under ultra-high vacuum, let alone interpret 

experimental observations of systems under industrial 

conditions. Hence, there is a need for theoretical models 

that can identify the most likely surface structures, and 

predict how they will react under realistic conditions in a 

complex environment. These models can then be used to 

guide experiment and interpret complex results. 

The new double-sinker 

densimeter developed by CSTL 

researchers defines the new 

best-in-the-world instrument for 

wide-ranging, high-accuracy P-ρT measurements. Furthermore, 

it provides an absolute measure 

of density, i.e., no calibration 

fluids are needed. 

Understanding and controlling 

surface chemistry of chromium 

oxide has a broad impact on 

the catalyst, automotive, steel, 

and aluminum manufacturing 

industries. 
33

We have developed a method of ab initio thermodynamics that enables us to link 0 K density 

functional theory electronic structure and vibrational calculations to finite temperatures and 

pressures to calculate the free energy of a surface in 

equilibrium with multiple species in the environment. 

The resultant phase diagram of the α-Cr2O3 (0001) 

surface in the presence of H2O, and O2 indicates a 

complex surface chemistry with an extreme sensitivity to 

environmental conditions, which can account for much of 

the variability and controversy in previous studies. Upon 

exposure to sufficient oxygen pressure, exposed Cr 

atoms on the surface react to form strong chromyl Cr=O 

bonds. This chromyl group is extremely stable and can 

explain how oxygen poisons the surface of chromia 

catalysts and passivates the surface of stainless steel, which is primarily chromium that has 

segregated to the surface. The calculated phase diagram also indicates that this chromyl group 

can be removed by low oxygen pressure and high temperature, which is consistent with 

experimental observations of catalyst regeneration and susceptibility to corrosion in stainless 

steel after exposure to heat and vacuum. 

Alcoa is very interested in using this technique to engineer surfaces of chromiumaluminum alloys. Hence, we will be extending this technique to calculate the 

phase diagrams for mixed-metal oxide surfaces and to identify processing 

conditions that favor formation of desirable surfaces. In addition, due to the great 

sensitivity of the chromium oxide surface structure and bonding to the environment, we plan to 

explore the potential of this oxide as a chemical sensor. 

Gas Chromatography Retention Index Library: A Major New Resource Nears Completion 

J.K. Klassen, P.J. Linstrom, and S.E. Stein (838) 

CSTL researchers have nearly completed a comprehensive gas 

phase retention index library for capillary columns. These data are 

abstracted from the literature and evaluated using structure-based 

predictive algorithms. Data entry and evaluation software has 

been developed specifically for this program, including specialized 

methods for selecting reference values to convert relative 

retention data to more useful retention index values. Estimation 

methods based on these data, along with various data comparison 

routines have been developed, tested, and employed for finding 

suspect values in the collection. 

The evaluated data collection will be provided along 

with the NIST Mass Spectral Library to confirm the 

identity of compounds in chemical analyses. These 

data will also provide a basis on which to build welltested predictive methods. These estimates can assist 

analysts in making identifications of compounds not 

represented in the Mass Spectral Library. Only 

nonpolar stationary phases are included in this first 

version, and polar phases will be added over the next 

year. Also, all relative retention times will be converted 

to retention indices by linking selected measurements 

to retention index values already in the collection. This 

collection will be regularly updated to reflect the latest 

This collection will assist in the 

confirmation of identifications made 

by matching acquired mass spectra 

against NIST library spectra in two 

ways. First, for those compounds 

with reference retention data, a check 

against the measured retention index 

will either improve the reliability of an 

identification or exclude a potential 

false positive result. For compounds 

not having measured values, this 

collection provides the basis for a 

structure-based estimation scheme. 

NISTís new Gas 

Chromatography 

Retention Index Library 

contains approximately 

70,000 data points for 

12,000 compounds on 

widely used nonpolar 

capillary columns. 

The phase diagram of the α-Cr2O3

(0001) surface in equilibrium with 

O2, H2O, has been calculated from 

first principles, and is consistent 

with experimental observations. 

Formation of a strong Cr=O bond 

upon exposure to oxygen can 

account for the passivation of 

stainless steel and oxygen 

poisoning of catalysts. 
34

values available in the literature. Both these data and estimation algorithms will be made 

available to users of the NIST Mass Spectral Library. 

High-Throughput Liquid Transport Properties Measurements in Membranes and Films 

C.D. Muzny, S.L. Outcalt, J.L. Scott, and L.A. Watts (838); B.J. Bauer and R.C. Hedden 

(MSEL) 

Measurements of liquid, vapor, and gas diffusion and sorption in thin layer films are critical for 

development of techniques to predict membrane transport properties. These measurements 

provide a means to compile the effects of both chemical and structural subgroups in the material, 

and ultimately, to delineate rational design criteria for mass separating agents such as 

membranes and polymeric adsorbents. A high-throughput apparatus for the measurement of the 

diffusivity and solubility of a variety of test components in 

a wide variety of membranes is under development. 

This technique employs an array of sixteen test cells that 

are individually temperature controlled and that can be 

loaded with differing solvents, test solutions and 

membranes under analysis. A critical component of this 

method is to have easily detectable, standard test 

compounds that probe different physical and chemical 

characteristics of the membranes. Toward this end a set 

of test fluorophores has been identified and produced in 

large quantities. Automation of the experiment, a key to any high-throughput method, is 

proceeding using the techniques of robotic sample delivery and fiber-optic sample detection 

systems. 

The selection of test fluorophores and their production in 

quantities large enough for the experiments under 

design was a major accomplishment. After testing for 

fluorescence properties and stability, G4 PAMAM 

dendrimers that have their sizes modified by the 

attachment of polyethylene glycol surface groups of 

various molecular weights were chosen as the key test 

compounds. These were then produced in gram 

quantities via size-exclusion chromatography (SEC) in a 

set of four different sizes, each fluorescently labeled with 

a different color for easy separation and detection. 

Tests of fluorescent response concentration linearity, 

solubility in water and ethanol, and size validation were 

performed. The automation of the high-throughput 

apparatus has been completed via the use of a robotic injection system. Cells with individual 

solvent flow control can have small quantities of test 

fluorophores injected via this robotic injector. Sample detection 

is then accomplished through fiber-optic connection to a highsensitivity fluorescence detector. A control system capable of 

simultaneously controlling the temperature and fluid flow on both 

sides of the transport cells is in place. Final system tests are 

currently underway. The availability of both the data and 

predictive methods will impact all industries that currently use 

membrane-based separations including the water purification 

industry, the pharmaceuticals industry, and the chemical 

industry. 

NIST researchers provide critical 

data for the characterization of 

membranes and films according 

to their species-specific 

transport properties. These data 

will then be available for use in 

the rational design of separation 

processes.

Although polymeric and inorganic 

materials are used in membrane and 

adsorptive separation processes, a 

significant barrier to the optimum 

use of existing materials and 

development of new materials is the 

lack of predictive capabilities for the 

transport properties of mixtures in 

any selected material. This data 

production effort will facilitate the 

development of the quantitative 

structure-property relationships 

necessary for the development of 

the predictive methods. 
35

Industrial Fluid Properties Simulation Challenge: Benchmark Standards 

D.G. Friend, J.W. Magee, A.M. Chaka, R.D. Mountain, R.D. Johnson III, L.A. Watts, E.W. 

Lemmon, R.D. Chirico, M.O. McLinden, A. Laesecke, and T.J. Bruno (838) 

 

The First Industrial Fluid Properties Challenge was sponsored by the Computational Molecular 

Science and Engineering Forum Group (CoMSEF Group) of the American Institute of Chemical 

Engineers (AIChE) in order to promote and to advance the status of molecular simulation for the 

calculation of properties of interest to industry. CSTL has had a key role in organizing the 

Industrial Fluid Properties Challenge, 

together with industrial participants from 

such companies as Dow Chemical, BP, 

DuPont, etc. In particular, NIST has 

coordinated activities related to 

establishing benchmark standards 

against which competition entries will be 

compared. Questions posed by the 

competition were prioritized by the 

industrial participants and included the 

calculation of certain vapor-liquid 

equilibrium properties, densities, and 

viscosities. A total of twelve supporting 

documents were created, involving about 

35 benchmark points with uncertainties. 

In some cases, the benchmarks were based on a careful evaluation of 

published or unpublished experimental results. For other challenge 

questions, experimental measurements were conducted for the density 

and viscosity of specified pure fluids and mixtures conducted at Dow 

Chemical and at NIST. The NIST measurements, in particular, required 

analysis and purification of available specimens, extension of the 

temperature/density range of a vibrating tube densimeter apparatus, 

and establishment of calibration points at high densities using a new 

double-sinker densimeter. 

Contest entries were evaluated in a double-blind protocol, 

and the announcement of results and discussion were held 

at a special symposium at the National AIChE Meeting in 

November 2002. 

Plans are being considered for a second challenge to be completed in 2004; experiences gleaned 

from the first competition, as well as suggestions from industry and practitioners will serve as 

input. The paths toward systematization of force field development and evaluation of algorithms 

will be identified as this project progresses. 

 

Molecular simulation is widely perceived to be 

a tool of the future in the determination of 

thermophysical properties of interest to 

industry. In some contexts, it is currently 

considered as an alternative to measurements 

or conventional theoretical approaches. 

However, this tool has limited value because of

the absence of a critical evaluation of its 

capabilities: no one knows how good the 

results are. A workshop on this subject was 

held at NIST in 2001, and the framework for the 

simulation challenge was established.

The simulation contest will serve 

to assist in the transfer of the 

technology to prospective 

industrial users, to identify 

strengths and weaknesses of the 

various techniques, to begin the 

process for systematic 

development of force fields, and 

to initiate general protocols for 

the assessment of property 

prediction techniques. 

Contest champions in three categories were selected: 

1. Andreas Klamt of COSMOlogic GmbH & Co.; 

2. Huai Sun of Aeon Technology, Inc; and 

3. Marcus Martin and Aidan Thompson from Sandia 

National Laboratories.
36

Measurements, Modeling and Data for Pressure-Driven Membrane Separations 

C.D. Muzny, K. Benko, and J.L. Scott (838); M. Chapman-Wilbert and K. Price (U.S. Bureau 

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

Analysis techniques for the standardization of membrane performance testing in pressure-driven 

separations are under development. These techniques include methods for the characterization 

of both the membrane and the complex mixture to which the membranes are subjected for 

separations testing. The membrane is characterized by a 

novel liquid-liquid porosimetry apparatus for the 

measurement of pore size distributions, while the standard 

fouling mixtures are characterized by field flow 

fractionation. By producing a standard fouling mixture that 

includes particulates and a range of large organic species, 

we are able to determine membrane performance under a 

variety of realistic conditions. These standard test 

solutions and procedures will provide industry with a 

uniform method for pressure-driven membrane separation 

performance testing. A reverse osmosis membrane 

module test bed is under development for the validation of 

the developed test solutions under realistic conditions. 

As part of this development, it is 

necessary to have standard 

techniques for the evaluation of 

both the membrane and the test 

solution. The membrane must be 

evaluated for chemical, physical, 

and structural characteristics 

important in the development of 

rational design criteria for 

separations for a wide variety of 

industries. A unique liquid-liquid porosimetry apparatus for physical 

characterization of membranes that we previously developed has 

undergone extensive testing during the past year. This testing has 

included membranes for ultrafiltration, nanofiltration, and reverse osmosis. 

The final validation of this technique is still underway, but preliminary 

results indicate it will be useful for membranes with pore sizes from 2 nm 

to 1000 nm. This includes virtually all membranes of current technological 

relevance. The characterization of the standard fouling solutions via field 

flow fractionation has continued. Validation and standardization methods 

for this technique have been developed in order to provide repeatable and 

reliable data for standard solution characterization. As a final test of the 

overall goal of providing standards for pressure-driven membrane 

performance testing, a small-scale membrane module test bed is under 

development. This will have the capability of testing the performance of 

spiral wound, reverse osmosis modules under the standard conditions 

being separately developed and characterized. The final production of a 

membrane test bed will be the major effort of next year. 

Researchers develop standard 

methods for the determination 

of membrane performance in 

pressure-driven separations. 

The ultimate goal is to provide 

a standard test mixture for 

testing membrane fouling 

along with standard 

procedures for evaluating 

membrane performance. 

Improved processes for obtaining 

specialty chemicals, 

pharmaceuticals, and advanced 

monomers using environmentally 

benign processes, and more 

economic ways to recover, reuse, 

and supply water are examples of 

important industrial and municipal 

uses of membranes. 
37

Notre Dame Radiation Laboratory (NDRL)/NIST Solution Kinetics Database on the Web 

T.C. Allison, R.E. Huie, and P. Neta (838)

The NDRL/NIST Solution Kinetics Database has been made accessible on the Internet at (NIST 

Standard Reference Database 40) and is one of the few databases with this type of information 

available. A powerful search engine is part of the 

web site allowing rapid searching of the database, 

and subsequent links to additional NIST databases 

will make this a particularly important source of 

information on free radical chemistry in solution to 

such areas as physiological and radiation chemistry.

The rates of free radical reactions in 

solution are important in many parts 

of chemistry and biology, particularly 

with the increased understanding of 

their role in the causes and cures of 

disease and in aging. 

The database was converted from an older 

format to one more appropriate for use in 

conjunction with a web environment, and a 

web site was established. The web site 

incorporates useful information and links in 

addition to the implementation of the 

database. A search engine was integrated 

into the web site allowing for fast and accurate 

searching of the database. Results of queries 

are returned in a useful summary form with 

access to full details available with a single 

click. All records are indexed such that data 

are intuitively linked (e.g., one can go directly 

from a single reaction record to all records 

reported within a journal article). 

Over the next fiscal year, the database will be linked to 

the NIST Chemical Kinetics Database (gas phase) and 

the NIST Chemistry WebBook and vice versa. This will 

provide maximum linking of similar data, which will lead 

to a more user-friendly experience for those using the 

database. A new tool for data entry will be developed to 

enable distributed data entry to facilitate new data 

projects. The first of these will be to add reactions of 

phenoxyl radicals to the database; alkoxyl radicals will 

follow these. In developing a new data entry tool and 

gathering new data, the types of information contained in 

the database will increase. 

The Solution Kinetics Database 

contains over 23,000 rate 

constants, primarily for free 

radical reactions. More than 

11,000 unique species in more 

than 150 solvents or mixtures of 

solvents are included. The data 

have been abstracted from nearly 

4,000 literature sources.

Next Steps: 

An expanded definition of 

database fields will be used as the 

basis for an XML schema for 

solution kinetics data. The web 

site will have the ability to return 

data in the XML format for further 

processing by the database users. 

This functionality will ultimately 

be implemented as a web service 

allowing automated queries of the 

database.

http://kinetics.nist.gov/solution
38

Photochemical Reduction of CO2 Catalyzed by Metalloporphyrins 

P. Neta (838); and J. Grodkowski (Institute of Nuclear Chemistry and Technology, Warsaw) 

The accumulation of CO2 in the atmosphere from fossil fuel burning leads to global warming. 

Various strategies are being considered to mitigate this problem. One of the more promising is to 

mimic nature and carry out the photochemical conversion of CO2 into useful products, a strategy 

of photochemical reduction. Reduction of CO2 can lead to the formation of various useful 

chemical compounds, such as CO, HCOOH, CH2O, CH3OH, and CH4, which can either be used 

as fuels or as chemical feedstocks. Iron and cobalt porphyrins and related metal complexes have 

been proposed as catalysts for the 

photochemical reduction of CO2. In order to 

successfully design production systems based 

on these catalysts, detailed fundamental 

information is needed on their photochemical 

and chemical properties, including their stability 

under operating conditions. Our approach has 

been to utilize them in model photochemical 

systems, to demonstrate formation of CO 

and/or HCOOH from CO2, and then to examine the mechanism of catalysis and to determine the 

relevant rate constants by pulse radiolysis. This fundamental investigation into the photochemical 

reduction of CO2 in the presence of metal porphyrins provides the Department of Energy with the 

information required for them to decide how and if process design based on homogeneous 

photocatalysis should go forward. 

These researchers have found that iron and cobalt 

porphyrins (MP) catalyze the photochemical reduction 

of CO2 to CO in dimethylformamide or acetonitrile 

solutions containing triethylamine (TEA) as a reductive 

quencher. In these photochemical studies, the 

quantum yields were low, and the yield can be 

dramatically increased by the use of p-terphenyl (TP) 

as a photosensitizer. TP is very effectively 

photoreduced by TEA which can reduce Co and Fe 

porphyrins very rapidly to their M0

P state. While this 

highly reduced state reacts with CO2 to form CO, it also 

reacts with protons (formed in the photolysis) leading to 

production of H2 and also to hydrogenation of the 

porphyrin. After the metalloporphyrin is hydrogenated 

in several successive steps, it decomposes and the 

catalytic reaction stops. 

Metal complexes of similar structures exhibit similar 

behavior. Attempts to protect the metal complexes 

against decomposition by embedding them in 

zeolytic materials did not succeed. It appears that 

photochemical reduction of CO2 with metal 

complexes as homogeneous catalysts suffers from 

the side reaction due to photochemically produced 

protons. A more promising route is to investigate 

heterogeneous systems using semiconductor 

nanoparticles as light absorbers and catalysts. 

Besides being more stable, such systems could lead to better charge separation, possibly leading 

to the simultaneous reduction of CO2 and the oxidation of water. 

CSTL researcher leads the development 

of kinetic and mechanistic information 

on the elementary reactions involved in 

the photochemical reduction of CO2 and 

provides strategies for the reduction of 

CO2 into fuel or feedstock materials. 

Although iron and cobalt porphyrins are 

able to serve as efficient homogeneous 

catalysts for the photochemical 

reduction of CO2, the decomposition of 

these substances has proven to be an 

intractable problem. Thus, we have 

recommended that there be no attempt 

to use this approach in subsequent 

process design. 

Photolysis time, h

0246

Yield, mmol L-1

0

1

2

3

4

CO

H2

Photolysis of iron porphyrin in 

acetonitrile solution containing 

triethylamine p-terphenyl and 

saturated with CO2
39

Properties and Processes for Cryogenic Refrigeration 

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

R. Gates and L. Reynolds (Univ. of Colorado); and J. Pfotenhauer (Univ. of Wisconsin)

The use of cryocoolers in many technological applications has been hampered because of 

problems such as: short lifetimes, inefficiency, high cost, and excessive vibration. The objectives 

of this research are (1) to use measurement and modeling techniques for evaluating and 

improving performance of cryocoolers and 

their components, such as heat exchangers 

and pulse tubes, (2) to develop new and 

improved refrigeration and heat transfer 

processes for the temperature range below 

about 230 K, and (3) to provide a database on 

material properties at cryogenic temperatures. 

A book chapter on the thermodynamics of 

regenerative refrigerators was completed and 

reviewed this year. The book chapter lays the 

foundation of thermodynamics for oscillating 

fluid systems such as Stirling, GiffordMcMahon, and pulse tube refrigerators, and will aid the entire cryocooler industry to improve the 

performance of their cryocoolers.

Development of our regenerator test apparatus has begun with components designed and 

fabricated. A prototype pulse tube neon liquefier to provide 500 W at 30 K was developed and 

tests performed on several geometry options. A low temperature of 30 K has been obtained, but 

for liquefaction of neon a no-load 

temperature of about 20 K is required. 

Further tests and data analysis are 

underway under a CRADA with an 

industrial sponsor. Under another CRADA 

a 15 K two-stage pulse tube refrigerator 

was designed for use with a thermoacoustic 

driver. We have assisted the sponsor in 

the fabrication and testing of the system. 

Under a third CRADA we have designed 

and have partially fabricated a test twostage pulse tube refrigerator for cooling 

superconducting motors and for comparisons with our models. A microscale heat exchanger of 

parallel plate geometry was optimized, developed, and tested for use in a Joule-Thomson 

cryocooler for the Air Force. We have added Youngís modulus of materials at cryogenic 

temperatures to our web 

site as well as a complete 

set of properties data for 

many new materials. 

Cryocoolers are required for many 

technology areas, including the cooling 

of infrared sensors for surveillance and 

atmospheric studies, the cooling of 

superconducting electronics, magnets, 

and power systems, the cooling of 

cryopumps for clean vacuums in 

semiconductor fabrication processes, 

the liquefaction of natural gas, and many 

other existing and potential applications. 

NIST researchers and collaborators have 

provided industry with new tools to improve the 

performance of cryocoolers, particularly those 

operating at temperatures below about 20 K. 

This work on two-stage pulse tubes at 30 K will 

aid the superconductor industry in the 

development of superconducting systems that 

can be marketed to a larger customer base. Our 

materials properties database is now getting 

more attention and is useful to all industries 

dealing with cryogenic temperatures. 

http://www.cryogenics.nist.gov/NewFiles/material_properties.html
40

The NIST Mass Spectral Library: A Major Update 

A. Mikaya, S.E. Stein, D.V. Tchekhovskoi, C.L. Clifton, and Q.-L. Pu (838) 

After four years of intensive evaluation and measurement, a new version of the NIST Mass 

Spectral Library has been completed and delivered to distributors. The number of spectra has 

increased by over one-third, with each spectrum 

subjected to a published evaluation method. A 

variety of quality improvements have been made 

throughout the library in which names were rechecked, edited and added. Also, 40,000 new 

CAS registry numbers were added to assist linkage 

with other chemical databases. A re-ranking of 

replicate spectra and 

replacement of lower 

quality with higher 

quality spectra has 

led to the replacement of thousands of spectra in 

the main library with higher quality spectra. These 

enhancements in coverage and quality will improve 

the reliability of this library for identifying unknown, volatile chemical 

compounds using the technique of gas chromatography/mass 

spectrometry (GC/MS). 

Spectra for over 54,000 compounds 

were added to the new version, for a 

total of 174,948 spectra of 147,198 

compounds. This includes large 

numbers of spectra specifically 

measured for the library, including 

derivatives (compounds volatilized 

through chemical reaction), chemical 

weapons related compounds, and 

pesticides as well as replicate 

measurements for selected compounds. 

Spectra for 1,735 commercially 

available compounds of special interest 

were measured by GC/MS at NIST. All 

new spectra were subjected to 

published quality control 

procedures. Moreover, 

a variety of quality 

improvements were 

made throughout the 

library. All chemical 

names were re-examined to discover errors and to select the best name. IUPAC 

names were generated by software from structures for approximately 100,000 compounds. 

The Mass Spectral Database is a 

comprehensive, reliable library of 

mass spectral ëfingerprintsí to assist 

the task of compound identification 

using the method of gas 

chromatography/mass spectrometry 

(GC/MS). Approximately 2,500 new 

libraries are installed on instruments 

each year.

0

200

400

600

800

1000

1200

0 20 40 60 80 100

Peaks/Spectrum

Spectra

NIST '98

NIST '02

Over 16,000 spectra formerly in the main library 

were replaced by either higher quality replicates 

or newly acquired spectra. This led to a 

significant change in the distribution of peaks per

spectra in the library, as illustrated in the figure. 

IUPAC
41

The NIST WebBook - NIST Chemical Reference Data for Industry 

P.J. Linstrom and W.G. Mallard (838) 

During 2002 the eighth edition of the NIST 

Chemistry WebBook was released. The 

WebBook remains one of the most used 

resources for chemical and physical property 

data. The fraction of returning users, typically 

between 45% to 55%, is a good indication that 

the user community feels that the resource is 

valuable. For the first time a predictive tool has 

been added to the WebBook. The wellestablished group additivity method for enthalpies of formation as well as heat capacity and 

entropy has been added as a tool for users who need data for which there are no experimental 

data. The group additivity tool has been developed using the most recent analysis of the data on 

the WebBook. Additional new fluids were added for the high-precision fluid properties section. 

Ongoing work on developing standard protocols 

for transmission of chemical data has included 

work with IUPAC and ASTM groups associated 

with developing standards for data transmission. 

Thermodynamics Research Center (TRC) Comprehensive Program on Critical Data 

Evaluation

M.L. Frenkel, R.D. Chirico, Q. Dong, G.R. Hardin, and R.A. Stevenson (838); V.V. Diky 

(Belarussian State University, Belarus); X. Yan and X. Hong (Contractors) 

Thermophysical property data evaluation is currently one of the most time-comsuming and 

resource-consuming stages of process design in a number of industries (chemical, petroleum, 

pharmaceutical, etc.). It has also very significant implications in the analysis of mutual relations 

of various properties within particular classes or groups of chemical substances, which is 

traditionally a subject of fundamental research. 

However, unprecedented growth of the 

thermophysical data becoming available at 

present (almost doubling every 10 years) 

makes it practically impossible to employ 

traditional (static) methods of data evaluation 

used over the last 50 years. The main objective 

of this project is to create new efficient methods 

and computer tools for the thermophysical, 

thermochemical, and transport property data 

collection, critical evaluation, mining, quality 

control, management, and dissemination and to 

provide high-quality recommended data in the 

forms directly suitable for industrial and 

scientific applications. 

The WebBook is being used by a very 

wide variety of users, between 10,000 and

20,000 per week, in industry, government 

and academia. Usage has continued to 

grow at about 20% per year with over 

500,000 distinct IP addresses accessing 

the WebBook in the last year. 

http://webbook.nist.gov/chemistry

The NIST Chemistry WebBook was voted the winner in the Portals and Information 

Hubs Category of the ìBest Chemical Sites on the Webî sponsored by: 

ChemIndustry.com, Inc; John Wiley and Sons, Inc; and the Royal Society of Chemistry

The establishment of the TRC Data Entry 

Facility and TRC Data Quality Assurance 

Program has dramatically increased TRC 

capabilities in volume and quality of the 

ërawí experimental data collecting, 

forming the necessary data management 

foundation for the implementation of the 

dynamic data evaluation concept. The 

use of the Guided Data Capture (GDC) at 

the TRC Data Entry Facility made it 

possible to establish a rate of data entry 

at 200,000 data points per year. 
42

The new concept of ìdynamicî data evaluation has been developed at TRC. This concept 

requires the development of large electronic databases capable of storing essentially all the 

ëraw/observedí experimental data known up-to-date with detailed descriptions of the relevant 

metadata and uncertainties. The combination of these electronic databases with artificial 

intellectual (expert-system) software designed to automatically generate recommended data 

based on available ëraw/observedí experimental data leads to the ability to produce data 

compilations dynamically or ëto order.í This concept contrasts sharply with static compilations, 

which must be initiated far in advance of need. Dynamic compilations dramatically reduce the 

effort and costs associated with anticipating future needs and keeping static evaluations current. 

The recommended 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. The TRC Data Entry Facility 

was established to support a mass-scale data entry operation for the TRC SOURCE data system. 

Guided Data Capture (GDC) software was developed at TRC and released from NIST in 2002 to 

enhance data entry operation both in-house and from the outside sources. The development of 

the ThermoML, XML-based standard has been completed, to promote interoperability in 

experimental thermophysical and thermochemical property data collection and management 

worldwide. The Data Quality Assurance Program has 

been designed to control the data quality at various 

stages of data entry, evaluation, and database 

management. Implementation of this program in 2002 

allowed TRC to establish the most complete and 

reliable data set of critical parameters of organic 

compounds. The TRC Consortiumís Eleventh Annual 

Workshop was the second hosted by NIST, and was 

held to inform the company members on the progress 

made during the past year. 

A Standard Chemical Identifier 

D.V. Tchekhovskoi and S.E. Stein (838) 

The objective of this program is to develop a means of generating a unique 

identifier for a chemical compound from a digital representation of its chemical 

structure. It is being developed as an IUPAC 

project with the intention of establishing it as a 

standard, international means of representing 

chemical identity. The structure of the identifier 

has been established. It has an extensible 

ìlayeredî structure so that different structural 

types appear in separate layers. Within an IUPAC committee, a 

variety of chemical rules have been developed and are now 

implemented as algorithms that can reliably treat most features 

of organic compounds. Extensions are underway for inorganic 

and organometallic compounds. 

Future plans include the 

establishment of a framework of 

direct data communication, quality 

assurance, processing, and 

distribution between major journals in 

the field and NIST, and the 

development of the software 

prototype to implement the Dynamic 

Data Evaluation Concept for phase 

equilibrium property groups. 

CSTL scientists have 

developed, tested, and 

implemented algorithms 

for extracting a unique 

chemical identifier from 

an input connection 

table representation of a 

chemical structure. 

IUPAC
43

The basic methods for construction of the Identifier 

have been finalized. It is expressed as a series of 

ëlayers,í each containing a specific class of structural 

information. The layers are: chemical formula, 

connectivity, isotopic, stereochemical, tautomeric, 

and charge. This modularization increases the 

robustness of the Identifier, permits the inclusion of 

all known structural features and, most importantly, 

enables extensibility. Creation of an Identifier is 

done in three steps: 

1) Normalization which applies various structural 

rules to the input structure to extract only that information required for chemical identification. 

A key simplifying feature is the elimination of all pi-bonds, radicals, and charges. All of the 

ëchemicalí rules are expressed in this step, including definitions of tautomerization (H-atom 

migration) and stereochemistry. 

2) Canonicalization generates a unique label for 

each unique atom in the normalized structure. This 

uses well-developed ëgraph theoreticalí methods. 

This information can serve as feedback to the 

chemist for error checking. 

3) Serialization transforms the ëcanonicalizedí 

structure into strings of characters, one string for 

each layer. These layers, together, constitute the 

identifier. 

Most of the features involved in the treatment of organic compounds have been implemented and 

tested. The algorithms were implemented in a test program that was distributed, on request, to 

well over 60 individuals, who represent a wide range of applications and fields of study. 

Comments from this testing, along with input from a variety of other source, are being used to 

complete the processing of organic compounds. 

An Efficient Model to Compute the Exact Exchange Potential in Density Functional Theory 

C.A. Gonzalez and T.C. Allison (838); E. Ludena, V. Karasiev, and I. Gonzalez (Instituto 

Venezolano de Investigaciones CientÌficas, Venezuela ); and Y. SimÛn-Manso (Universidad 

de Chile) 

The use of state-of-the-art electronic structure calculations based on ab initio quantum chemical 

methodologies has become widely popular. Today, scientists in industry and academia make use 

of this technology to guide complicated experimental work in a large variety of areas including 

chemical reactivity, catalysis, chemical kinetics, combustion chemistry, environmental chemistry, 

and organic synthesis. Density functional theory (DFT) 

has become one of the most widely used 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 

ìexchangeî energy term and a ìcorrelationî energy term. 

Although, in principle, there exists a universal DFT 

functional that can describe the electronic problem 

exactly, the discovery of such functional has eluded scientists around the world 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. Systematic validation 

conducted by the Computational Chemistry Group of CSTL indicates that most of these 

functionals are not transferable to the computation of different chemical properties, where they 

The Identifier provides a standard 

means of communicating and 

storing the chemical identity of a 

substance from a structural 

drawing. Since no such standard 

now exists, this could greatly 

facilitate the digital communication 

of chemistry in a wide range of 

applications involving the storage 

and dissemination of chemical data.

Next Steps: Over the next year, the 

identifier will be extended to inorganic 

and organometallic compounds and 

distributed to a number of 

organizations for testing. Ultimately, 

the means for producing the identifier 

from a chemical structure will be 

distributed by IUPAC for integration 

into chemical processing software. 

CSTL researchers and 

collaborators have developed 

and implemented a robust and 

computationally efficient 

exchange functional for 

density functional theory.
44

show a marked erratic behavior. The exchange functional for density functional theory developed 

under CSTL leadership is computed variationally through the standard Kohn-Sham procedure 

and does not require parameters. It is shown that this functional (and its potential) reproduces 

the ìexact exchangeî behavior and that it eliminates serious problems such as lack of selfinteraction correction, non-variational character, and existence of spurious electron correlation, 

encountered in most of the exchange functionals used currently by the scientific community. 

The results of the work presented here indicate that the major source of error can be traced back 

to the ìexchangeî functional. It has been found that these functionals are non-variational and do 

not eliminate serious self-interactions of the electrons. In 

addition, the parameterization used to generate these 

functionals includes spurious electron ìcorrelation.î In this 

work, we have developed a method called ìSelf-Consistent 

Alphaî (SC-α) that eliminates all these problems and does not 

introduce further parameterization. The SC-α method 

involves the correction of the DFT exchange energy and its 

corresponding potential at each Kohm-Sham cycle by a factor 

computed as the ratio between the Hartree-Fock exchange 

and the DFT exchange. 

The use of the SC-α methodology will allow scientists to compute physical and chemical 

properties of complicated chemical systems in a reliable and cost-effective manner. The 

development of this methodology constitutes a major advancement in the area of DFT, a 

computational chemistry technique widely used by scientists in industry. 

The SC-α method has been 

successfully implemented and 

tested on a series of atoms 

and diatomic molecules for 

which values for the ìexact 

exchangeî energies are 

available. The results show 

excellent agreement between 

the SC-α values and the ìexact

exchangeî results. 

Future Plans: Further work leading to validation of the SC-α method in the 

calculation of different physical and chemical properties of polyatomic 

systems is under way. In particular, properties such as potential energy 

barriers critical for kinetic studies of chemical reactions will be studied. A 

similar study in order to improve the current correlation functionals will also 

be conducted.
45

Bruno, T.J. and Lagalante, A.F., “Process for the 

Removal of COS from LPG,” United States Patent 

6,334,949, Jan 1, 2002. 

Bruno, T.J. and Rybowiak, M.C., “Method and

Apparatus for Entrainment Mixing of Vapor into

Liquids,” (patent pending). 

5. Energy Systems 

Many CSTL projects impact this industry sector (electric power 

generation, oil, petroleum and coal, and natural gas) from reference 

data and models to 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. 

Chemical Characterization and Properties of Trace Components of Fuel Gas 

T.J. Bruno and W.C. Andersen (838); and A. Abdulagatov (Dagestan Scientific Center of 

Russian Academy of Sciences)

The U.S. consumes approximately 23 trillion cubic feet of fuel gas each year. Natural gas 

consists primarily of methane, but 400 additional compounds may be present at varying levels. 

Most of these materials are naturally occurring (heavier hydrocarbons, sulfur compounds, water, 

carbon dioxide, etc.), and some are added during 

processing. Liquefied petroleum gas (LPG) 

consists primarily of propane, but many other 

compounds are present. The efficient, safe 

design of equipment operated with fuel gases 

critically depends upon the properties and 

chemistry of individual components of natural 

gas and LPG. Moreover, 

it has become very clear 

that the components 

present at minor levels 

(or even at trace quantities) can have a significant impact on the overall 

properties of the fuel gas mixture. 

Recent work has included (1) completion of work on carbonyl sulfide 

(COS) hydrolysis in propane, (2) the relationship of COS hydrolysis on 

the Copper Strip Corrosion Test (CSCT), and (3) a comprehensive 

evaluation of the applicability of the CSCT. Kinetics measurements 

revealed that the hydrolysis reaction of COS is too slow to be of 

consequence for the propane industry. Measurements were made at 

several temperatures, and in the presence of nine catalysts. An Arrhenius plot was developed to 

provide a predictive approach to the hydrolysis kinetics. In addition, we studied the effect of 

hydrocarbon diluents in this multiphase reaction system. We determined that the hydrocarbon 

imposes a diffusion barrier to the reaction, and that a correlation exists between the diffusion 

coefficient of the hydrocarbon and the reaction rate constant. These kinetics measurements were 

confirmed with CSCT measurements done on COS 

mixtures in propane. This evaluation of the CSCT 

revealed significant operational problems with the test, 

and we have taken steps to improve the test. 

Energy Systems

Researchers provide the natural gas 

industry with chemical characterization 

and properties that are critically needed 

for the efficient operation of fuel gas 

processing, transmission, and 

distribution. This is required for 

custody transfer, calorific value, quality 

control, safety, and environmental 

compliance.

CSTL work on COS hydrolysis 

and its relation to the CSCT will 

change the way the propane 

industry deals with this sulfur 

contaminant. The comprehensive 

review of COS chemistry will 

replace the earlier 1957 review, 

and the technology developed 

during this project will be used in 

industry and the laboratory. 
46

Thermal Conductivity of Natural Gas Constituents 

R.A. Perkins (838) 

Natural gas and liquefied petroleum gas are abundant and clean burning fuels that typically 

consist of mixtures of light hydrocarbons with nitrogen and carbon dioxide. Knowledge of the 

thermal conductivity of natural-gas mixtures is required to optimize heat exchange surfaces in 

processes for power conversion, liquefaction, 

separation, and purification. This work 

focuses on the thermal conductivity of pure 

propane, butane, and isobutane at 

temperatures from their triple points to 600 K 

at pressures up to 70 MPa. New transient 

hot-wire measurements of thermal 

conductivity cover the vapor, liquid, and 

supercritical gas phases with reduced 

uncertainty and fill regions where data were not available. The thermal conductivity data are 

used to develop accurate correlation surfaces for these three pure fluids that form the basis of 

improved models and standards for natural-gas mixtures. These data revealed errors in the best 

available correlations for thermal conductivity that were greater that 10 % near the critical density 

of each fluid and again approaching the triple point of butane and isobutane. These new data 

were used to develop reliable models for the thermal conductivity of these important constituents 

of natural-gas mixtures. In particular, propane is an ideal reference fluid for extended 

corresponding states models of the transport properties of natural gas mixtures since its liquid 

state exists over an exceptionally wide range of reduced temperature. 

Natural gas and liquefied petroleum gas are multicomponent blends with a wide variation in 

composition. Corresponding states models are applicable to ternary and higher-order mixtures 

and allow a complete description of the thermal 

conductivity of natural gas and liquefied petroleum gas 

mixtures. The improved knowledge of the thermal 

conductivity of propane, butane, and isobutane enables 

the development of better thermal conductivity models 

for these fuel mixtures. In addition, these three gases 

are considered to be “natural refrigerants” and their use 

has been proposed in alternative refrigerant blends. 

These improved models for thermal conductivity have 

been incorporated into the recently released Version 7.0 

of the NIST REFPROP database. 

Release of Version 7.0 of the NIST REFPROP Database

E.W. Lemmon, M.O. McLinden, and M.L. Huber (838)

Industry requires high-accuracy thermophysical 

property data for the pure fluids and fluid mixtures 

used as working fluids in refrigeration and air 

conditioning systems. Property databases are 

widely needed for the design and analysis of 

refrigeration systems and are also used for other 

applications such as flow metering, calibrations, 

and the design, optimization, and innovation of 

chemical processes. A major revision of the NIST 

REFPROP database has been completed and is 

now being distributed by the NIST Standard 

Reference Data Program. Previous versions of 

CSTL researcher provides the natural gas 

industry with high-accuracy 

thermophysical property measurements 

and reference standard models for natural 

gas constituents and their mixtures, over 

wide ranges of temperature, pressure, and 

composition.

The improved models described 

here were developed in 

collaboration with the 

International Association for 

Transport Properties and are 

recommended as interim 

standards. IATP was formerly 

known as the Subcommittee on 

Transport Properties of IUPAC. 
47

The thermophysical properties of 

light hydrocarbons of between 5 

and 8 carbons make this family of 

compounds attractive as heat 

transfer fluids for solar panels used 

in electric power generation. 

this database have become the de facto standard 

for refrigerant properties worldwide. The new 

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. Support is provided for using the property 

routines with other applications, including Excel 

spreadsheets and Visual Basic programs. 

Version 7.0 represents a major revision and 

upgrade of this database. New features include additional fluids and mixtures. The revision adds 

the main air constituents nitrogen, oxygen, and argon, and thus, REFPROP 7.0 supplants the 

NIST 72 database on the properties of air. Ammonia/water mixtures are also included, 

incorporating a model that had been previously planned as a separate database. Arbitrary 

mixtures of up to 20 components may be specified, up from the five-component maximum in 

previous versions. Properties of HFC mixtures are calculated with a new Helmholtz energy 

model, and experimentally based values of the mixture parameters in this model are now 

available for 145 binary pairs. The viscosity and thermal conductivity models for 19 fluids have 

been updated based on recently published data. The new graphical user interface (GUI) retains 

the overall look and feel of the Version 6 GUI, but 

incorporates many enhancements and new features and 

option with greatly improved speed of calculation, and 

many more options to prepare plots of thermodynamic 

surfaces. Additional properties, including dielectric 

constant, freezing lines, and sublimation lines have been 

added where data are available. A wide variety of 

property diagrams may be automatically generated, 

including pressure-enthalpy and temperature-entropy 

diagrams and (for binary mixtures) temperaturecomposition and pressure-composition plots. 

Stability of Potential High-Temperature Heat Transfer Fluids 

D.G. Friend, T.J. Bruno, and W.C. Andersen (838) 

 

In this solar energy application, heat transfer fluids 

are subjected to high temperatures and pressures on 

the order of 315 °C and 4.1 MPa (600 °F and 600 

psia). At these temperatures and pressures, the 

stability of many such fluids is uncertain, especially 

long term, and in contact with potentially catalytic 

surfaces. We are working with the National 

Renewable Energy Laboratory (NREL) to establish 

the best values for key thermophysical properties of the 

pentanes and, especially, to determine the stability of 

pentanes and light aromatics. We are also measuring 

the kinetics of decomposition at 315 °C and 4.1 MPa in 

the presence and absence of catalytic materials. The 

experimental results, based on reaction chromatography 

and ampoule decomposition kinetics measurements, 

indicate that these fluids may not be sufficiently stable for 

the intended application. 

Industrial users now have at their 

fingertips the most accurate 

properties available with the updated 

database entitled Reference Fluid 

Thermodynamic and Transport 

Properties (REFPROP, Version 7.0). 

High-accuracy equations of state for 

pure fluids and their mixtures have 

been implemented along with models 

for the transport properties. 

Previous versions of REFPROP 

have become the de facto standard 

for refrigerant properties. The 

REFPROP subroutines have been 

embedded in many industrial and 

commercial applications and 

provide consistent property values. 

Version 7.0 provides additional 

capabilities, increased accuracies, 

and data for additional pure fluids 

and mixtures. 
48

There has been growing interest in integrating parabolic trough solar systems with organic 

Rankine power cycles (ORC); this integration of technologies would open a new market niche 

where solar technologies have been too expensive. Although ORC technology is very mature, 

the existing plants operate at temperatures below about 200 °C. Preliminary analysis at NREL 

showed that pentanes (and fluids with similar thermophysical properties) as working fluids could 

provide high cycle efficiencies. Unfortunately, these fluids had not been tested at high 

temperatures, and there were concerns about decomposition of the working fluids. NIST was 

asked to respond to concerns raised by the solar and ORC industry to assist in determining the 

suitability of pentane isomers in parabolic trough solar systems by study of the stability of the 

fluids at high temperatures with and without catalytic materials. We have completed our initial 

efforts to establish key properties for the pentane isomers to be used in cycle design calculations. 

The stability measurements are based on two techniques: (1) reaction chromatography, a method 

developed at NIST (see illustration), and (2) ampoule decomposition kinetics measurements. 

These methods allow the reaction to be measured as a function of temperature in the presence 

and absence of catalytic surface materials. Results for n-pentane, for example (with residence 

reaction times up to about 10 days), indicate a rate constant for decomposition of about 8.4 x 10-5

min-1. Isopentane, neopentane, and toluene have also been shown to decompose at the 

conditions of interest. In conjunction with researchers at NREL, several additional heat transfer 

fluids and mixtures have been identified. Uncatalyzed reaction screening measurements will be 

performed on two candidates, and continued study depends, in part, on results from these 

experiments. 

Properties of Refrigerant Mixtures Near the Critical Point 

M.O. McLinden, R.A. Perkins, E.W. Lemmon, A. Laesecke, and J.W. Magee (838) 

The refrigerant blends now seeing commercial application as replacements for HCFC-22 in airconditioning equipment can operate near the refrigerant critical point. This exacts a performance 

penalty, and this project is supplying improved property data and models in support of the 

equipment testing and simulation work being 

carried out in BFRL to elucidate the causes of the 

performance penalty. Measurements of heat 

capacity, density, viscosity, and thermal 

conductivity have been completed. It has also 

resulted in significant advances in our equation-ofstate models and fitting procedures. These results 

have improved the accuracy of our refrigerant 

mixture models, which are transferred to industry 

primarily through the NIST REFPROP database. 

The results to date indicate that 

most of the common fluids 

examined are not suitable as 

heat transfer fluids in solar 

trough applications. Thus, 

these fluids will not be studied 

further in laboratory scale or 

pilot demonstration facilities. 

However, the integrated 

technology is still of interest, 

and alternative heat transfer 

fluids are being considered. 

Increasingly, refrigeration systems 

are using refrigerant blends, and the 

new models developed here have 

improved the accuracy of HFC 

mixture properties. These improved 

models have been incorporated into 

the recently released Version 7.0 of 

the NIST REFPROP database. 
49

HCFC-22 is the refrigerant presently used in most residential and small 

commercial air-conditioners and heat pumps, but it must be phased out 

under international treaty. Refrigerant mixtures are the likely replacement, 

and systems using mixtures are now on the market. These blends, typically 

composed of HFCs 32, 125, 134a, and/or 143a, have much lower critical 

temperatures than HCFC-22 and normally operate at considerably higher 

pressures. Under extreme conditions, the blends can operate near or 

above the refrigerant critical temperature and pressure, with major losses in 

capacity and efficiency. An understanding of the causes of such losses is a 

high priority of the Air-Conditioning and Refrigeration Technology Institute’s 

industry-sponsored 21-CR research program. There are very few property 

data for the blends near their critical points. This work has focused on the 

measurement of new data for R-410A (a blend of HFC-32 and HFC-125) 

near its critical temperature. The new data, together with 

other available measurements for HFC mixtures, have 

been used to develop improved models for the entire 

class of HFC blends over wide ranges of conditions. In 

the course of this effort, significant advances in our 

modeling and fitting procedures have been made. 

These include nonlinear fitting techniques and 

theoretical constraints, resulting in models with fewer 

parameters that are yet more accurate with improved 

extrapolation behavior. 

Viscosity Measurements and Model Comparisons for Second-Generation Alternative 

Refrigerant Blends 

A. Laesecke (838) 

Significant energy penalties have been measured in HVAC systems with next-generation 

alternative refrigerant blends operating at condenser temperatures that are closer to their critical 

temperatures. To understand the causes of these energy penalties, working fluids have to be 

characterized not only at subcritical saturated vapor and 

liquid states but also at supercritical conditions. 

Literature viscosity data for such blends and working 

conditions are very sparse. Accordingly narrow is the 

basis on which predictive viscosity models for such 

mixtures have been developed. Also, substantial errors 

have been identified frequently in literature viscosity data 

for hydrofluorocarbon systems. Therefore, additional 

viscosity measurements are needed to build a broader 

base of validated and reliable data for these important 

fluids. 

Measurements on the blend R410A in two different viscometers presented an opportunity to 

discern the susceptibility of this fluid to an applied electric field and to quantify its effect on the 

results of the torsional crystal viscometer in an effort to extend the application range of this 

instrument. Wide-ranging viscosity measurements of the blends R507A (0.5 R143a + 0.5 R125 

by mass) and R410A (0.5 R32 + 0.5 R125 by mass) were carried out in the torsional crystal 

viscometer at two subcritical and three supercritical isotherms with pressures up to 68 MPa. The 

subcritical measurements were conducted at the nominal temperatures 300 K (liquid only) and 

340 K (liquid and vapor). Supercritical measurements included the nominal temperatures 360 K, 

390 K, and 420 K. For reference, the viscosity of the blend R410A was measured at saturated 

The measured viscosity data 

fill large gaps for R507A and 

R410A alternative refrigerant 

blends. They serve as a 

reference to examine literature 

data for measurement errors, 

and they give indications 

where the predictive model 

may be improved. 

The new model calculates all 

thermodynamic properties in the 

liquid, vapor, and supercritical 

regions with typical uncertainties of 

0.1% in density and 0.5% in heat 

capacities and the speed of sound. 

Calculations of the bubble or dew 

point pressure along the vaporliquid equilibrium boundary have 

uncertainties of 0.5%. 
50

liquid conditions between 240 K and 350 K with a sealed gravitational capillary viscometer 

because in that instrument the sample is not exposed to an electric field. The measurement 

results were compared with literature data and with values estimated via the extended 

corresponding states model in NIST Standard Reference Database 23 (REFPROP, Version 7). 

Besides their industrial relevance, the 

results of this study laid the foundation for 

the development of the torsional crystal 

viscometer to probe flows under 

combined molecular and electroviscous 

friction as occur frequently in microfluidic 

systems. 

 

100 (η exp /η REFPROP - 1)

-8

-4

0

4

8

12

16

0 200 400 600 800 1000 1200 1400

ρ / kg∙m-3

0.752 R125 by mass 0.50 R125 by mass 0.241 R125 by mass

Nabizadeh, Mayinger (1999) Fröba et al. (2001)

Heide, Schenk (1996):

This work:

R507A (0.50 R125 by mass):

300 K 340 K 360 K 390 K 420 K

R143a + R125
51

6. Environmental Technologies 

This large industry sector 

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 decisionmaking for environmental remediation, waste disposal, 

emissions trading and other applications. 

SRMs for Contaminants on Air Particulate Matter 

S.A. Wise, R. Oflaz, J.R. Kucklick, B.J. Porter, D.L. Poster, M.M Schantz, & R.L. Zeisler 

(839) 

The Environmental Protection Agency (EPA) issued new standards in 1997 for air 

particulate matter (PM) under the national ambient air quality standards. These 

standards added new regulations for PM less than 2.5 :m (PM2.5), which is the 

respirable PM fraction, in addition to the existing PM10 standards. Research 

recommendations have already 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 materials are 

currently available to 

support this research. 

CSTL is collaborating 

with EPA to provide SRMs and quality assurance tools for measurements 

of inorganic and organic species in PM2.5. 

Newly released SRM 2783 air particulate on filter media, provides 0.5 mg 

of fine air particulate matter on a polycarbonate filter membrane with 

certified mass loadings for 24 toxic and environmentally important 

elements. This SRM is intended for elemental analysis using XRF, 

Neutron Activation Analysis, and other analytical 

techniques. For organic compounds on PM2.5, we 

are in the process of collecting a 200-g bulk 

sample of ultra-fine particulate matter at a site in 

Baltimore, MD. This PM collection will be 

completed in late 2003, and the material will be 

used for preparation of a bulk PM2.5 SRM with 

values assigned primarily for organic 

constituents. Because the collection, 

preparation, and certification of this PM2.5 SRM 

will require 2 to 3 years, an interim reference 

material was prepared from a 20-g batch of PM2.5

To expand the quality assurance 

capabilities, NIST has entered an 

agreement with the US-EPA to develop, 

certify, and issue a suite of SRMs that 

will be used for the evaluation of 

analytical methods to provide national 

measurement traceability and to 

improve interlaboratory comparability 

of measurements of today’s PM. 

The interim reference material has been 

distributed to 18 laboratories as part of a 

NIST/EPA interlaboratory comparison exercise 

for the determination of organic compounds in 

fine particulate matter. The results of this 

interlaboratory study will be used in 

conjunction with NIST measurements to assign 

concentration values for the compounds of 

interest and will be available to laboratories 

involved in EPA PM2.5 research programs for 

use as a control or reference material. 

Air Sampling Station

The highly fragmented U.S. 

environmental industry is 

large and complex 

encompassing four primary 

segments: water supply and 

treatment, solid waste 

management, air pollution 

control and environmental 

cleanup. The global market for

environmental technologies is

estimated to be $530billion.
52

Gravimetry quantified the variation in 

loading that is observed here. An 

intercomparison exercise was 

designed to utilize accepted thermaloptical analysis protocols to explore 

possible loading effects. 

collected earlier at the Baltimore site during different sampling periods in 1998-99 and 2001. This 

interim reference material has been characterized at NIST for polycyclic aromatic hydrocarbons 

(PAHs) and nitro-substituted PAHs. Measurements are in progress for other classes of 

compounds that are of interest because of toxicity and/or as source markers including: alkanes, 

alkenes, hopanes, sterols, aldehydes, ketones, phenols, acids, and inorganic species. 

Gravimetric Results of a Fine Particulate Carbonaceous Reference Material on QuartzFiber Filter 

G.A. Klouda (837) and J.J. Filliben (ITL) 

Gravimetric results have been compiled and analyzed for the production of RM 2784 urban dust 

on quartz-fiber filters, including process and control blank filters. RM 2784 is on a quartz-fiber 

filter and is specifically intended for carbon analysis. 

Bulk carbon measurements, organic vs. elemental 

carbon, rely on thermal techniques and therefore 

require a non-carbonaceous filter. Beyond the need to 

assign values of elemental carbon (EC), organic carbon 

(OC), and total carbon (TC) for each method, the 

results of this exercise are expected to yield information 

regarding possible mass interval effects, within-filter 

homogeneity, and filter-to-filter homogeneity. 

A randomization plan was designed to set protocols for 

loading and handling of the filters, and the production of 

process blank and control blank filters. The distribution 

of PM loadings and relationships with respect to chamber position, chamber and batch was 

variable: average mass loading and standard deviation for 2231 filters was (1064 ± 414) μg 

PM2.5 per filter with a range of 2763 μg and a minimum and maximum of 92 μg and 2855 μg, 

respectively. Given the relative standard deviation of mass loading of 39 %, filters identified for a 

round-robin exercise to assign values for EC, OC, and TC will be randomly selected from eight 

stratified mass-loading intervals. Two of the most 

widely used thermal-optical methods, STN and 

IMPROVE, will be the focus with two of the most 

qualified laboratories representing each method. The 

design will be balanced with a strong emphasis on 

replication and true round-robin sampling. 

RM 2784 has been designed and 

produced especially for the 

seven EPA “Supersites” that are 

monitoring fine airborne PM 

throughout the U.S. 

 CSTL researchers have 

determined mass loadings for 

individual filters produced by a 

particle re-suspension system. 

RM 2784 will provide the means to intercompare 

methods and laboratories for the measurement of 

elemental carbon in PM. Use of this RM will 

determine the level of agreement between sites and 

establish traceability to stated references based on 

thermal-optical techniques. 
53

TOA response surface maps; 

lower map is rotated highresolution view of upper map 

Chemometric Optimization of Thermal-optical Analysis in Determining Black Carbon in 

Atmospheric PM2.5

J.M. Conny, D.B. Klinedinst, S.A. Wight, and J.L. Paulsen (837) 

Atmospheric black carbon (BC), a ubiquitous component in fine (<2.5 μm diameter) particulate 

matter (PM2.5), is responsible for most of the uncertainty in climate change prediction. The 

chemical, physical, and morphological complexity of aerosol BC presents major problems in 

assuring measurement accuracy. PM2.5 

metrology including BC accuracy is also a 

major concern of industrial leaders currently 

addressing the new EPA PM2.5 emissions 

standard. Methods based on thermal-optical 

analysis (TOA) are widely used for ambient air 

samples; however, different TOA thermal 

desorption protocols result in wide BC to total 

carbon (BC/TC) variation. We created three 

surface models of the following response 

variables from TOA: BC/TC, maximum laser 

attenuation in the He phase (Lmax), and laser 

attenuation at the end of the He phase (LHe4). A two-level 

central-composite factorial design comprised of four factors 

considered the temperatures and durations of all desorption 

steps in TOA’s inert (He) phase and the initial step in TOA’s 

oxidizing (O2-He) phase. The Lmax surface was used to 

optimize the production of pyrolized organic carbon (OC char) 

in the He phase. Unpyrolized OC is measured as native BC, 

causing a positive bias. The LHe4 surface was used to 

minimize the loss of char in the He phase, which served as a 

surrogate indicator for loss of native BC in the He phase, a 

source of negative bias. The intersection at the Lmax and LHe4

surface maxima revealed conditions that minimize potential 

biases, leading to an optimized thermal desorption protocol. 

Environmental Scanning Electron Microscope (ESEM) images 

were used to better understand changes in particle population 

morphology during the TOA process. 

 

We plan to apply these chemometric techniques to thermaloptical analysis of several U.S. regional PM2.5 samples. This 

will help to fine-tune the optimized thermal desorption protocol 

from this work, differentiate it if necessary for different types of 

ambient air samples, and validate robust TOA operational 

protocols. 

CSTL scientists improve the 

understanding of the key operational 

variables in thermal-optical analysis 

(TOA) by a systematic and repeatable 

chemometric approach. This is needed 

by the black carbon measurement 

community to improve intercomparability 

and interpretation of results based on 

measurement accuracy. 

This quantitative map of the TOA response 

surface will help scientists to derive the most 

appropriate instrumental conditions for accurately 

measuring black carbon by TOA. 
54

Total Carbon Determination in Buffalo River Sediment Using Isotope Dilution Mass 

Spectrometry: Comparison of Techniques

D.B. Klinedinst, R.M. Verkouteren (837); and K. Revesz (U.S. Geological Survey)

Accurate measurements of carbon are of great relevance to many 

sectors of the U.S. economy, and carbon is rapidly becoming an 

economic global commodity 

due to its tremendous 

relevance in global climate 

change. Unfortunately, there 

are no SRMs with certified

carbon concentration values. 

While non-certified values 

exist, they have generally 

relied on operationally dependent measurement 

techniques that are frequently referenced to standards 

with mismatched matrices. Isotope dilution mass 

spectrometry (IDMS) is a well-known and “absolute” 

technique for determining the concentration of a 

particular analyte, and is especially applicable for 

complex matrices. IDMS concentration measurements depend entirely on isotope ratio 

measurements of a sample, a labeled spike material, and an equilibrated mixture of the two. 

Quantitative recovery of the analyte is not required. 

Here, we utilized 13C-depleted urea and determined 

mixing ratios with gravimetry. We have now 

developed, tested, and compared two IDMS 

methods for carbon using RM 8704 (Buffalo River 

Sediment). The resulting carbon values are 

consistent and comparable, but with uncertainties 

that require further development of the method. 

Upon successful completion of this project, we will 

certify high-priority SRMs for carbon to validate 

carbon sequestration methods in soils. In the near 

future, these may be used to establish carbon 

“credits” that offset greenhouse gas emissions. 

Mass Determination of a Population of Asbestos based on Particle Size and Number 

J.R. Verkouteren (837); and A.G. Wylie (Univ. of MD) 

The different agencies regulate asbestos on the basis of either mass or particle size/number, 

however it is clear that a more cohesive approach is needed. In addition, a much lower threshold 

limit for mass is being considered that is below the 

detection limit of the standard methods. The new 

technique described here provides a critical link between 

particle number and mass and provides the basis for a 

new method for determining low concentrations of 

asbestos in bulk materials. 

NIST and USGS researchers 

investigate a definitive method 

for certification of carbon in 

SRMs. This capability is critical 

for the Nation to meet upcoming 

challenges in carbon 

sequestration, and to provide 

accurate benchmarking 

materials for many industrial 

sectors concerned with the 

carbon content of feedstocks 

and process residues. 

The carbon concentration of RM 8704 

(Buffalo River Sediment) was 

determined with IDMS using urea 

depleted in 13C as an internal standard. 

Isotopic ratios were measured using 

two isotope ratio mass spectrometry 

techniques: dual inlet/IRMS (NIST) and 

Gas Chromatography/IRMS (USGS). 

The mass fraction of carbon in RM 8704 

was 0.03279 (0.0079 uc, n=3) and 0.0320 

(0.0124 ui, n=1), respectively. The 

relative difference between the existing 

RM 8704 carbon reference value and the 

new NIST result was 1.6%. 

Researchers address a 

critical measurement issue, 

which will likely result in new 

regulations for asbestos. 
55

In this work, the total particle mass of a population of asbestos 

fibers was determined from 2-dimensional measurements of 

length and width. A known mass of asbestos was deposited on a 

filter, and 50 randomly selected fields were imaged on the fieldemission-gun scanning electron microscope (SEM) at three 

different magnifications. The fiber lengths and widths were 

measured, and the thickness calculated by assuming one-half 

the measured width. The fibers were binned by length, width, 

and mass and the total number in each bin over the entire filter 

was calculated. Overlap in the frequency of bins common to two 

magnifications indicated internal consistency in the calculation of 

total number. The frequency distributions were modeled by a 

power series, consistent with the fractal model proposed for 

asbestos. The total mass was calculated both by summing the 

particles in each mass bin, and by integration of the fitted 

distribution. Although there are very few particles in the large 

mass bin that contributes greatly to the total mass, their number 

is predicted correctly by the modeled distribution. Although we 

have not yet determined the uncertainty of the final mass 

calculation, the values arrived at 

by the two different calculation are 

quite close to the weighed value, 

lending support to the overall 

approach. This approach should 

lend itself to accurate measures 

of mass concentrations at 0.1%, 

which is the new threshold limit 

being discussed. Without such a 

method, many new materials might be labeled as asbestoscontaining based on the discovery of a few fibers and the inability to convert this correctly to mass 

concentrations. 

SRMs for Contaminants in Soils, Sediments and Tissues

C. Beck II, T.A. Butler, R.R. Greenberg, M. Howard, W.R. Kelly, J.R. Kucklick, R.M. 

Lindstrom, S.E. Long, E.A. Mackey, J.L. Mann, A.F. Marlow, K.E. Murphy, R. Oflaz, R.L. 

Paul, B.J. Porter, D.L. Poster, M.M. Schantz, J.R. Sieber, G.C. Turk, R.D. Vocke, S.A. Wise, 

L.J. Wood, L.L. Yu, R.L. Zeisler (839); and S. Tutschku (Universitat Leipzig, Germany) 

Validation of the complete analytical method for environmental measurements requires the use of 

natural matrix SRMs similar to those matrices typically encountered in the analysis of 

environmental samples. NIST natural-matrix SRMs for organic and inorganic contaminants in 

soil, sediment, botanicals, and tissue are necessary for the development and validation of 

analytical procedures and to provide quality 

assurance of chemical measurements for these 

samples. The environmental community, including 

those responsible for promulgating analytical 

methods, developing policy, enforcing regulations, 

and interpreting laboratory data, has long known 

that risk to biota and humans is dependent upon the form of the metal in the environment not just 

the total elemental composition. SRMs with values assigned for speciated metals are required to 

address these measurements needs. 

The long-term impact of 

this work is to provide a 

new framework for 

asbestos regulations that 

relates the hazardous 

material (defined in terms 

of mass) with the risk 

(defined in terms of 

particle size/number). 

Expectations are that species-based 

regulation is imminent despite the 

uncertainties in the underpinning 

measurement science. 

log mass, mg

-12 -10 -8 -6 -4 -2 0

log frequency -1

0

1

2

3

4

5

6

7

SEM fiber image and mass 

distribution of an asbestos 

fiber population

143 μm 
56

Recent soil, sediment, botanical, and tissue matrix SRMs have been developed to address this 

need and include: marine sediment (SRM 1941b and SRM 2702), mussel (SRM 1974b) and fish 

tissue (SRM 1946), pine needles (SRM 1575a), and mine waste-contaminated soil (SRM 2780). 

SRM 1941b Organics in Marine Sediment and SRM 1974b Organics in Mussel Tissue are 

renewals of these popular marine matrices for determination of organic contaminants with 

certified values for over 60 constituents. SRM 1575a Pine Needles (a renewal), SRM 2702 

Inorganics in Marine Sediment, and SRM 2680 

Hard Rock Mine Waste are intended for the 

determination of elemental composition. SRM 

1946 Lake Superior Fish Tissue is the first 

frozen fish tissue SRM and is intended primarily 

for the determination of contaminants such as 

polychlorinated biphenyls (PCBs), chlorinated 

pesticides, total mercury, and methylmercury, 

as well as food matrix components such as 

proximates and fatty acids. The marine 

sediment, fish, and mussel tissue SRMs were 

developed to support the numerous laboratories 

involved in marine environmental monitoring. 

Another area of concern is the environmental impact of 

mine waste drainage on the neighboring communities. 

There are an estimated 500,000 abandoned mines in 

the western U.S., and these sites commonly contain 

significant quantities of discarded mine waste, resulting 

in acidic drainage containing elevated levels of heavy 

metals. Accurately evaluating the environmental impact 

of mine waste drainage has been hampered by the 

variability observed in laboratory methodology used to 

assess this hazard. The mine waste SRM was 

developed to support these measurements. 

In addition, in cooperation with the EPA, the New Jersey Department of Environmental 

Protection, and USGS, NIST has begun development of a new SRM to address speciated forms 

of metals, primarily chromium-VI, in contaminated soils. A contaminated soil known to contain 

Cr-VI has been collected and is being prepared by USGS for use as the candidate SRM. In 

addition, a second fish tissue (SRM 1947 Lake Michigan Fish Tissue) has been prepared and 

measurements are in progress to provide a fish tissue SRM with emphasis on the trace element 

content to complement SRM 1946, which focuses primarily on the organochlorine contaminants. 

The stock material used for SRM 2702 is also being used to develop the first reference material 

(SRM 2703) for solid sampling direct analysis techniques that commonly use small analytical test 

portions. 

To address the need for reference materials 

for speciated metals, NIST has provided 

certified concentrations for methylmercury as 

well as total mercury in the two marine tissue 

SRMs (SRM 1946 and SRM 1974b) to 

complement the existing suite of mussel and 

oyster tissue SRMs with certified values for 

methylmercury (SRM 2976, SRM 2977, and 

SRM 1566b). Reference values were also 

provided for the renewal marine sediment, 

SRM 1941b, for mono-, di-, and tributyltin 

species in addition to total tin concentration. 

Abandoned Mine
57

Developing an Advanced Primary Ozone Photometer - A Collaboration with BIPM 

P.M Chu, J.E. Norris, and F.R. Guenther (839) 

The concentration of ozone in the atmosphere remains a significant issue from both scientific and 

political perspectives. Ozone, at tropospheric levels, is a health concern and contributes to global 

climate change as a greenhouse gas, while stratospheric ozone protects earth from harmful UV 

radiation. NIST has supported the ozone 

monitoring communities for many years 

with its Standard Reference Photometers 

(SRPs), however, increasing demands in 

both the national and international arena, 

require a new approach. Therefore, the 

current research effort is focused on 

coupling state-of-the-art measurement 

technologies with national primary gas 

standards to provide an advanced primary 

ozone standard, which will improve SItraceability and comparability of global 

ozone measurements. 

NIST/CSTL supports the growing national and international need of improved ambient ozone 

measurements by partnering with BIPM to develop advanced primary ozone reference standards 

and to share the dissemination of secondary or transfer standards. NIST/BIPM partnership 

follows includes: 1) The transfer the current ozone “national reference photometer” technology to 

BIPM. 2) Performance assessment of the current technology with respect to current and future 

ozone measurement requirements. 3) Development of a primary reference standard with 

capabilities of SI-traceability verification. 

During FY 2002, two new SRPs were built, 

extensively compared to the principal NIST 

reference photometer (SRP 2), and transferred to 

BIPM. A 0.5 % calibration bias between SRP 2 

and the SRPs transferred to BIPM has been noted 

and an ongoing effort is dedicated towards 

identifying and eliminating this bias. To assess 

the current comparability of global ozone 

measurements, a pilot ozone key comparison was 

initiated in the fall of 2002. An advanced 

photometer demands instrumentation with high 

sensitivity, accuracy, and precision, as well as 

rapid time response for measuring chemical 

species at atmospheric levels (nmol/mol and 

lower). A number of in-situ techniques are being 

considered, including time-gated laser-induced 

fluorescence detection, laser absorption 

spectroscopy, and cavity ring-down spectroscopy 

for detection of relevant chemical species and 

possible contaminants in the carrier gas. Collaborations with 

researchers developing state-of-the-art instrumentation to 

measure NO2, NO, and O3 at trace levels have been 

established. During the next year, two additional SRPs will be 

built for BIPM with participation of BIPM staff to accelerate the 

transfer of the current technology. Evaluation of potential 

instrumentation for the advanced primary reference standard 

will also be initiated. 

Since 1983, NIST has provided Standard 

Reference Photometers (SRPs) based on 

UV photometry to ten US Environmental 

Protection Agency (EPA) facilities to 

provide an infrastructure for the calibration 

and traceability of ozone measurements 

within the US. More recently, the 

international interest in ozone 

measurements has prompted fifteen 

foreign laboratories to acquire SRPs. 

The advanced primary ozone 

photometer will provide an 

internationally validated 

ozone standard that can be 

used to anchor all ozone 

measurements through a 

defined traceability structure. 

14 Mar 21 Mar 28 Mar 21 Apr 28 Apr 5 May

0.996

0.997

0.998

0.999

1.000

1.001

1.002

1.003

1.004

Slopes

 SRP28 vs SRP27 at NIST

 SRP0 vs SRP27 at NIST

 SRP28 vs SRP27 at BIPM

 SRP0 vs SRP27 at BIPM

14 Mar 21 Mar 28 Mar 21 Apr 28 Apr 5 May

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

Intercepets (ppb)

date

Comparison of NIST-constructed 

Standard Reference Photometers for 

ozone measurements: SRP 0 at NIST and 

SRPs 27 and 28 now located at BIPM
58

Standards to Support Stack Emissions Testing 

W.D. Dorko (839) 

The Clean Air Act was originally passed in 1977, and from that 

time through the 1980’s the traceability requirements were met 

by NIST developing Standard Reference Materials (SRMs). 

NIST developed technical specifications for the Specialty Gas 

Industry (SGI) to produce required gas mixtures, developed 

primary standards, and analyzed each of the candidate SRM 

mixtures for certification purposes. These SRMs were then 

made available for sale to form the traceability path for emission 

measurements. In 1990 the Clean Air Act was amended and 

part of the new regulation required vastly increased monitoring of the emissions, which required 

an order of magnitude increase in the usage of calibration gases. The 1990 Amendments also 

included arrangements for emissions trading whereby a 

power plant that emitted less than their “allowable” amount 

of SO2 could sell a permit to other companies for the 

balance. Measurements made against NIST traceable 

calibration gases are the foundation for emissions trading. 

Both the emissions trading and the increased monitoring 

required greatly increased usage of traceable gas 

standards. NIST did not have sufficient resources to meet 

this increase within in the context of the SRM program in 

which NIST invested all of the resources, and performed 

100% of the certification analyses. Therefore, NIST 

worked with the SGI and EPA to develop a new program, 

the NIST Traceable Reference Material (NTRM). In the NTRM program, the SGI invests most of 

the resources and NIST performs about 10% of the analyses for certification. The NIST 

resources are greatly leveraged with the NTRM program, and the SRM program is maintained. 

Since the inception of the NTRM program in 1992 more than a dozen companies have produced 

and NIST has certified multiple batches of NTRMs. Most of the batches consist of binary 

mixtures with air or nitrogen being the diluent gas. The analytes are varying concentrations of 

propane, methane, carbon monoxide, carbon dioxide, oxygen, hydrogen sulfide, nitric oxide, 

nitrogen dioxide, and sulfur dioxide. The demand for NIST traceable calibration gases cannot 

even be met with the NTRM program so EPA developed a document describing how Specialty 

Gas Companies (SGC) could produce “EPA Protocol Gases” that EPA accepts as NIST 

traceable. The procedure requires that the SGC analyze the Protocol Mixture against either an 

SRM or an NTRM. This is a mechanism to leverage NTRMs since one NTRM can be used to 

analyze 100 or more EPA Protocol mixtures. The results are that stack emissions have been 

maintained at the desired regulated levels, and the desirable ambient air quality has been 

reached. It has also resulted in a smooth functioning emissions trading program whereby 

companies that introduce new technology are rewarded and can sell the excess permits to emit to 

companies that have not yet capitalized for 

plant improvement. NIST is currently working to 

develop calibration gas mixture standards for 

chlorine, hydrogen chloride, and ammonia. The 

magnitude of the demand for these will not be 

as large as for the other gases so other 

traceability mechanisms are being explored 

outside of the existing NTRM and SRM 

programs. 

Stack gas emissions are 

regulated by the U.S. EPA 

under stipulations in the Clean 

Air Act. In order to quality 

assure compliance the 

instrumentation used to 

monitor these emissions must 

be calibrated with gas mixture 

calibration standards traceable 

to NIST.

Another Approach – Remote Sensing: 

NIST is also developing an infrared 

database to calibrate infrared-based 

sensors that analyze the emissions from 

stacks. If this is successful then it will be 

an alternate method to provide traceability 

for emissions measurements. 
59

Benchmark Data on Liquid Fire Suppressants 

C. Presser (836); J.F. Widmann (BFRL); G. Papadopoulos (Dantec Dynamics); and 

P. DesJardin (Sandia National Laboratory) 

The new generation of non-ozone-depleting Halon alternatives include chemical suppressants 

that have high boiling point temperatures (i.e., Tb > 330 K) and exist in the liquid phase under 

high-pressure release or in ambient conditions. Release 

of these agents in a confined cluttered space results in 

the dispersal of droplets that either travel along ballistic 

trajectories, move with the convecting flow, or impact 

nearby solid obstacles. Therefore, an accurate 

representation of agent droplet transport is crucial for the 

numerical modeling of fire suppression in confined 

spaces. To better understand the physics of droplet transport around and behind solid objects, an 

experiment was designed where controlled grid-generated turbulence was imposed on the air 

stream. Results are obtained using particle 

image velocimetry, phase Doppler 

interferometry, and visualization techniques. 

The liquid agents considered in the 

investigation were water, and two 3MTM fire 

protection fluorocarbons HFE-7100 (with a 

boiling point of 334 K), and HFE-7000 (with a 

boiling point of 307 K). 

Particle image velocimetry (PIV) experiments 

were completed with the assistance of Dantec 

Dynamics to characterize the impingement and 

transport of a water spray around a cylinder and 

body-centered cube arrangement of spheres 

that represents a more complicate obstacle. 

Measurements upstream and downstream of a 

33 mm cylinder having a 250 W heater and 

thermocouples to monitor the near surface 

temperature (see figure) were completed. The 

cylinder was heated to 423 K to study the 

effects of a heated surface on droplet 

vaporization and transport, as a droplet 

approaches the heated surface. The results 

indicated that the transport of droplets around 

the cylinder is highly dependant on droplet size, 

and spray impingement and surface wetting 

caused significant cooling of the heated 

surface. 

Plans are underway to initiate measurements with the phase Doppler interferometry system and 

to obtain spatial profiles of the droplet size and 

velocity distributions, and droplet number density 

upstream and downstream of the obstacles for water, 

HFE-7100, and HFE-7000. 

NIST scientists develop a 

parametric data set to guide and 

validate the Vulcan fire 

modeling efforts underway at 

Sandia National Laboratory. 

Understanding the correlation of the 

agent/spray properties, agent 

atomization methods, and agent 

dispersion effectiveness will enable the 

optimization of fire suppression 

performance of misted liquid systems. 

Experimental results provide new data 

for a well-characterized, homogenous 

droplet-laden turbulent flow field around 

prescribed obstacles 
60

Distribution of Mercury in Seabird Eggs as a Marker of Environmental Contamination in 

Alaska 

S.J. Christopher, P.R. Becker, S. Vander-Pol, R. Pugh, and R.D. Day (839) 

High latitude areas (Arctic and Sub-Arctic) tend to act as a sink for global pollutants such as 

mercury (Hg). NIST is collaborating with the U.S. Fish and Wildlife Service’s Alaska Maritime 

National Wildlife Refuge (AMNWR) and the U. S. Geological Survey’s Biological Resources 

Division, in the Seabird Tissue Archival Monitoring Project (STAMP). This project is using 

Alaskan colonial seabirds as markers to monitor the geographic and temporal trends of 

contaminants in the Alaskan marine environment. 

The purpose of STAMP is to monitor long-term 

trends in marine environmental quality through the 

use of colonial seabirds as indicators. NIST’s role in 

STAMP is to provide the quality assurance 

infrastructure of analytical specimen banking through 

the National Biomonitoring Specimen Bank and 

chemical analysis of contaminants in seabird tissues, 

including eggs, for STAMP’s collaborating agencies. 

Specimen banking and analytical protocols 

for seabird eggs were developed and 

published. Samples from 41 egg 

specimens from 4 different locations were 

analyzed for total mercury. Mercury 

signatures in common murre eggs were 

readily observed. Hg levels in the eggs 

were significantly different in the Bering Sea 

and Gulf of Alaska (see figure), indicating 

that regionally dependent environmental 

processes may produce conditions that 

allow for favorable uptake of Hg in Alaskan 

seabirds residing in the Gulf of Alaska. 

Cutting-edge analytical methods and accurate 

environmental Hg data produced at NIST were 

exported to our program sponsors. This research 

also yields benefits to the numerous researchers 

studying global Hg cycling and tracking Hg 

deposition in Alaska. The published Hg data were 

also provided to researchers in Alaska 

conducting risk assessments of subsistence 

foods on Alaska Native communities (murre eggs 

are consumed by local residents) and the data 

generated by NIST were a recent contribution to 

the international Arctic Monitoring and 

Assessment Programme. 

 

NIST provides support to the 

expanding STAMP program through 

the banking of seabird eggs in the 

National Biomonitoring Specimen 

Bank and egg contents analyses. 

Recent analyses of common murre 

(Uria aalge) eggs show the 

existence of regionally unique Hg 

chemical signatures. 

N=11 N=9 N=11 N=10

Mercury content (mean ± 95% C.I.) in 41 

common murre eggs plotted as a function 

of island colony and water body. 

Projected sampling sites 

and species for STAMP 
61

DoE SPR Well Head

Development and Critical Evaluation of a New ID/ICP-MS Method for Mercury in Sediments 

and Fossil Fuels 

S.E. Long and W.R. Kelly (839) 

A method combining high-pressure closed-system sample digestion with isotope dilution coldvapor generation inductively coupled plasma – mass spectrometry (ID-CV-ICP-MS) has been 

developed for the determination of mercury in environmental matrices. Refinements to the 

method, the most important of which has been a 

dramatic reduction in the magnitude of the 

procedural blank, now permit accurate 

determinations to be made at ultra-trace (pg/g) 

concentrations in any sample matrix. The method is 

being exploited to make measurements of mercury 

in complex matrices with much lower expanded 

uncertainties than previously attainable, and at 

concentration levels that cannot be matched by 

other techniques or methods. The development 

and implementation of this method fulfils an urgent 

need for more accurate measurements of mercury in environmental materials. Data provided by 

the method result in a much improved traceability link for industrial EPA compliance monitoring 

and for calculating anthropogenic mercury releases for risk assessment studies. CSTL will 

continue to work with EPA, DoE and other interested parties to ensure that these new and unique 

measurement capabilities for mercury analysis are fully exploited. One application that we are 

promoting is a study of mercury levels in crude oil feedstocks that are more representative of 

current refinery processing in North America. These data could be used to assess the 

contribution of mercury from these sources to the U.S. mercury budget. To date, this has not 

been assessed with any great reliance, owing to the 

limitations of existing analytical methods. The provision of a 

more accurate database will be useful for EPA regulatory 

decision-making and for strategic planning by the oil industry. 

A new analytical method has been 

applied to the determination of 

mercury in sediment, coal, and in 

crude oils representative of the 

Strategic Petroleum Reserve (SPR) 

stockpiles. Sales of NIST fossil fuel 

SRMs have dramatically increased 

following the addition of certified 

mercury data using this method. 

This work resulted in: 

• Dramatic reduction in the magnitude of the method 

process blank, now permitting accurate 

measurements as low as 50 pg/g of sample matrix 

• Certification of mercury in several NIST SRMs: 

• SRM 2693 Coal 

• SRM 2702 Inorganic Sediment 

• SRM 2721 Moisture and Sulfur in Crude Oil 

• SRM 2722 Moisture and Sulfur in Crude Oil 

• Determination of mercury in crude oil samples from 

the Strategic Petroleum Reserve (SPR) in support of 

the DoE 
62

Development and Critical Evaluation of New Methods for Determination of Methylmercury 

and Butyltin Species in Marine Tissues and Sediment 

M.M. Schantz, S.A. Wise (839), and S. Tutschku (Universitat Leipzig, Germany) 

An improved method was developed for the determination of methylmercury and butyltin 

compounds in marine sediment and tissue using microwave-assisted acid extraction or digestion 

and solid phase microextraction (SPME) followed 

by analysis using gas chromatography with 

microwave induced plasma atomic emission 

spectrometric detection (GC-MIP-AES). Using the 

SPME-GC-MIP-AES method, enrichment factors 

for methylmercury and butyltin compounds of 50 to 

100 were achieved compared to the typical hexane 

extraction, and measurements in marine tissue 

and sediment matrices were possible at 1 :g/kg to 

2 :g/kg (methylmercury) and 10 ng/kg to 100 ng/kg 

(butyltins). The SPME-GC-MIP-AES method was 

validated using several marine sediment and tissue matrix Certified Reference Materials (CRMs) 

with certified values for methylmercury and butyltin compounds. The SPME-GC-MIP-AES 

method was used to measure methylmercury in four marine tissue SRMs ranging from oyster 

tissue at (13.0 ∀ 1.0) :g/kg to fish tissue at (397 ∀ 13) :g/kg as Hg dry mass. Results from the 

SPME-GC-MIP-AES method were used in conjunction with results from NIST’s instrumental 

neutron activation analysis (INAA) as well as 

results from two other laboratories using different 

analytical techniques to assign certified values for 

methylmercury in oyster, mussel, and fish tissue 

CRMs. Mono-, di-, and tributyltin were measured 

in three sediment SRMs at concentration levels of 

(0.08 ∀ 0.03) mg/kg to (0.35 ∀ 0.05) mg/kg as Sn 

dry mass. 

The SPME-GC-MIP-AES method for methylmercury will be implemented for other candidate 

marine tissue SRMs including SRM 1974b Organics in Mussel Tissue and SRM 1947 Lake 

Michigan Fish Tissue. The method for butyltins will also be used to provide values for marine 

tissue and sediment SRMs. 

A suite of five marine tissue SRMs is now 

available with certified methylmercury 

concentrations ranging from (13.2 ∀ 0.7) 

:g/kg dry mass to (1380 ∀ 18) :g/kg dry 

mass. From the lowest certified 

concentration for any tissue CRM to the 

fish tissue SRM with a concentration just 

below an action level of 500 :g/kg wet 

mass, this suite of SRMs will provide the 

tools necessary to improve the accuracy 

and reliability of methylmercury 

measurements in the marine 

environment.

The importance of the determination 

of organometal species, such as 

methylmercury and tributyltin, is now 

widely recognized because of the 

high toxicity to humans and biota 

exhibited by these compounds. SRMs 

with values assigned for organometals are required to address these 

measurement needs. 

NEW SRMS AVAILABLE: 

SRM 1566b Oyster Tissue 

SRM 2977 Mussel Tissue 

SRM 1946 Lake Superior Fish Tissue
63

Measurement Quality Assurance Programs for Contaminants in Environmental Samples 

M.M. Schantz, J.R. Kucklick, D.L. Poster, S.J. Christopher, and S.A. Wise (839) 

Environmental monitoring programs sponsored by the National Oceanic and Atmospheric 

Administration (NOAA) and the Environmental Protection Agency (EPA) rely on contaminant 

measurements performed by numerous laboratories and therefore need to assess the quality and 

comparability of the analytical measurements from the various laboratories. CSTL’s Analytical 

Chemistry Division currently coordinates three quality assurance programs for contaminants in 

environmental samples. 

 

The NIST Intercomparison Program for Organic 

Contaminants in the Marine Environment started 

in 1987 with partial support until 1999 from 

NOAA and the EPA. Since 1999 this program 

has continued on a pay-to-participate basis with 

over 30 laboratories participating in the annual 

intercomparison exercises for organic 

contaminants in sediment and marine tissues. 

The NIST/NOAA Intercomparison Exercise for 

Organochlorines and Trace Elements in Marine 

Mammal Tissues was established with NOAA 

support in the early 1990’s with limited interlaboratory comparison exercises; however, in 1999 

the program was formalized with annual 

intercomparison exercises for both 

organic and inorganic contaminants with 

over 40 laboratories participating. The 

NIST/EPA Intercomparison Program for 

Organic Contaminants in PM2.5 Air 

Particulate Matter started in 2000 within 

the Particulate Matter (PM) 2.5 Organic 

Speciation Working Group sponsored by 

EPA and has currently has 25 laboratories 

participating in two interlaboratory studies 

for organic constituents in air particulate 

matter. Through each of these programs, 

NIST provides mechanisms for assessing 

the interlaboratory and temporal 

comparability of data and for improving 

measurements for the monitoring of 

organic contaminants in bivalve, fish, and 

marine mammal tissues; sediment; and 

air particulate samples and monitoring 

trace elements in marine mammal tissues. 

Currently over 75 laboratories participate in these 

three QA programs for environmental contaminant 

measurements. Because candidate SRMs are 

often used as the unknown materials in these 

exercises, the results are often used in conjunction 

with NIST measurements as part of the value 

assignment process for the SRM. 

Laboratory measurement accuracy has 

improved by participation in NIST’s QA 

exercises, resulting in sound 

environmental decision-making based 

on analytical measurements of 

assessed quality and comparability. 

Participation in these exercises is often 

a requirement for laboratories involved 

in federal and state government 

monitoring programs. 

SRM 1941b Organics in Marine Sediment, 

SRM 1946 Lake Superior Fish Tissue, and 

SRM 1974b Organics in Mussel Tissue 

were all used in QA exercises in recent 

years and the consensus results from the 

study were used in assigning the certified 

concentrations for various contaminants. 

0

10

20

30

40

50

60

70

80

90

100

mussel tissue sediment

1 year

2-5 years

6-10 years

11- 14 years

Years of

participation of

reporting lab

Percent of participant-reported analyte results within 

50% of the exercise material’s assigned value

Improvement in the accuracy of a lab’s measurement capabilities has been shown to correlate 

with the number of years of participation. 
64

Structure, Adsorptive Separations, and Characterization of Surfactant/Clay Complexes 

T.J. Bruno and K.E. Miller (838); and F. Tsvetkov (Daren Laboratories, Israel) 

Clays are used in a broad array of applications ranging from pollution prevention and remediation, 

enhanced oil recovery, the treatment of petroleum liquids, the manufacture of cosmetics and 

pharmaceuticals, and the synthesis of polymer nanocomposite materials. They are also a critical 

part of the land environment, and as such, must 

be considered in any pollutant-soil interaction. 

An understanding of clay-organic chemical 

interactions and the effects these interactions 

have on the structure of clay complexes is a 

critical issue for future developments in the 

environment and in clay applications. In order to 

understand the physical chemistry (both 

thermodynamics and kinetics) of the clay-solute 

interaction, we have advanced the application 

of physicochemical gas chromatography by devising stable clay- and organoclay-coated capillary 

columns. The capillary column approach we have developed is more efficient, requires lower 

temperatures, and produces enthalpy of adsorption ()Hads) results with a much lower uncertainty 

than the conventional techniques. 

We have completed the production and characterization of the first successful organo-clay 

coatings for capillary column gas chromatography. We used them to measure the enthalpy of 

adsorption ()Hads) for a family of hydrocarbons on a synthetic layered silicate complexed with 

cetyltrimethylammonium bromide (CTAB), and also on a synthetic layered silicate coated with 

octadecane. These substrates are especially significant as surrogate soil systems that can be 

used to model the interaction of pollutants on soils. In this work, we demonstrated that the )Hads

is uniformly lower for organoclay than it is for the pure clay surface, and concluded that the 

organic component "ties up" the most active sites on the clay. As an application of the work with 

clay and organoclay substrates, we studied the )Hads of fuel gas odorants on soil surrogates. This 

was motivated by the observation of odorant fading in fuel 

gas, a suspected cause of which is soil adsorption. We 

have derived the )Hads difference (∗)Hads) between fuel gas 

components and their respective odorants, and have used 

this value to predict fading. Our values of )Hads complement 

breakthrough values that have been measured on real soil 

samples. A significant result is the observation of a high 

)Hads for tetrahydrothiophene (THT), and a relatively low 

)Hads for tertiary-butyl mercaptan (TBM). We extended the 

work to hydrated clay surfaces and found that the )Hads is 

reduced for thiol odorants, an observation confirmed by 

breakthrough experiments. 

CSTL researchers exploit the unique 

characteristics of clay platelets in order 

to produce organic-inorganic 

composite materials with revolutionary 

material properties, and to develop of 

novel chemical separation. They have 

also used clays as a platform to study 

the pollutant-soil interaction.

An immediate impact of the 

NIST work has been the 

recognition of differential 

adsorption of the various fuel 

gas odorants. The (very 

common) presence of clay in a 

pipeline or the leak of a pipe up 

through a clay soil can have 

devastating consequences. Our 

results will allow avoidance of 

unfavorable odorants, and 

thereby improve safety. 
65

Molecular Simulation of Solute Diffusion (Sodium Chloride) in Supercritical Steam 

A.H. Harvey, and R.D. Mountain (838); and J.C. Bellows (Siemens Westinghouse) 

A large fraction of electric power is generated in 

facilities that employ steam turbines, either in the 

primary power cycle or as a secondary heatrecovery cycle following a combustion turbine. 

Essentially no experimental measurements exist 

for the diffusivity of solutes in supercritical steam, 

despite the importance of these data for 

understanding deposition in steam turbines and 

for design of supercritical water oxidation 

processes. In this work, molecular dynamics 

simulation was used to calculate the diffusion 

coefficient of the sodium chloride ion pair at 

infinite dilution in supercritical steam. Potentials 

from the literature were used; a polarizable model for the water molecule was chosen to 

accurately represent water in the vapor phase. 

The results of the simulations are estimated to be 

accurate to within 20%, which is a great 

improvement over current semiempirical 

estimation methods. The results can be fitted 

satisfactorily to the form of a simple kinetictheory expression, allowing extrapolation to 

conditions other than those simulated. The 

results from this work now provide the best 

values available for the diffusivity of sodium 

chloride in steam. 

Our approach is to calculate the diffusivity directly from molecular dynamics simulation, which 

provides rigorous results if the intermolecular potentials are sufficiently accurate. Good potentials 

(incorporating polarizability, which is needed for accurate 

description of water in the vapor phase) are available for 

water with sodium chloride. Advances in computing power 

have brought this calculation into the range of feasibility. 

Collaborations with the CSTL’s Computational Chemistry 

Group are planned to calculate reliable thermochemical 

data for hydrated silica species and to determine which 

species will need to be incorporated in our simulations. 

Kinetics and Thermophysical Properties of Ionic Liquid Systems 

P. Neta, J.W. Magee, M.L. Frenkel, A. Laesecke, M.O. McLinden, and R.A. Perkins (838); 

J. Grodkowski (Institute of Nuclear Chemistry and Technology, Warsaw); and G. Kabo 

(Belarusian State Univ., Belarus) 

Ionic liquids are a class of organic salts that are liquid at or near room temperature. They are 

generally composed of a large asymmetric organic cation and either an organic or inorganic 

anion. As such, they are nonvolatile and nonflammable. Therefore, they have been proposed as 

solvents for “Green Processing.” The physical properties of ionic liquids may be tuned, for 

example, by altering the hydrophobicity, and this affects the solubility, reaction rate, and 

selectivity. However, in spite of the many advantages that these fluids are predicted to offer, 

fundamental data on their physical and chemical properties are scarce. A physical properties 

database is being developed at NIST that will provide industry with access to evaluated physical 

CSTL researchers provide the first 

reliable data for the diffusion coefficient 

of a solute in steam at turbine 

conditions. Industrial water treatment 

experts have estimated that costs to the 

electric power generation industry of 

efforts to prevent and repair damage 

due to these impurities amount to 

hundreds of millions of dollars per year.

Steam Turbine Power Generator 

Redding Electric Utility 

This project is one of a series 

aimed at evaluating the utility 

of computer simulation 

methods for problems for 

which experimental data are 

not available. 
66

property data, validated models to interpolate 

the available measurements, and structureproperty tools to predict physical properties 

not yet measured. Measurements of physical 

property data are being collected for 

properties including melting point, density, 

electrical conductivity, refractive index, 

viscosity, heat capacity and surface tension. 

The focus of this data collection is on ionic 

liquid compounds that are liquid-phase at 

room temperature and are air- and moisture-stable, since they will have many practical 

applications. 

Though all possible sources are under consideration to uncover 

both published and unpublished measurements, it is becoming 

clear that measurements are limited for most physical properties 

and are nearly nonexistent for vapor pressure or thermal 

conductivity. A single compound, 1-butyl-3-methylimidazolium 

hexafluorophosphate, has been systematically studied, but not its 

vapor pressure or thermal conductivity. We have initiated 

experimental measurements with a focus on five physical properties for six high-purity 

compounds – density, heat capacity, vapor pressure, viscosity, and thermal conductivity. In our 

labs, we are measuring densities with a pycnometer, viscosities with Ubbelohde-type capillary 

viscometers, and thermal conductivities with a transient hot-wire apparatus. 

 

The pulse radiolysis technique was utilized to measure rate constants for several oxidation, 

reduction, addition, and hydrogen abstraction reactions in ionic liquids. In addition to the higher 

viscosity, significantly lower rate constants for redox reactions indicate that the ionic liquids 

behave as solvents of low polarity, suggesting a high degree of ion-association. Certain 

reduction and oxidation reactions are found to behave very differently in ionic liquids as compared 

with other solvents, both in the rate of the reaction and sometimes even in the direction in which 

the electron is transferred. These strong 

effects were found to be due to 

differences in the energy of solvation of 

the reactants and products in the various 

solvents, which affect the driving force of 

the reaction and thus its rate constant. In 

the future, the use of ionic liquids for the 

preparation and stabilization of colloidal 

metal nanoparticles will be explored. 

Ionic liquids, a class of organic salts that 

are liquid at room temperature, have been

proposed as solvents for “Green 

Processing.” To provide U.S. industry 

with the knowledge base to exploit these 

solvents, CSTL researchers have begun 

to study the physical properties of ionic 

liquids and their effects on rate constants 

of fundamental chemical reactions. 

A preliminary study has 

shown that the choice of 

either the cation or the 

anion strongly influences 

the physical properties of 

the ionic liquid.

In most cases the rate constants in ionic liquids 

are lower than those in classical solvents, in part 

because of the higher viscosities of ionic liquids, 

which limits the diffusion rate. 

 

Hydrogen abstraction and addition reactions, in 

general, are only slightly slower in ionic liquids 

compared with water and organic solvents. 
67

7. Food and Nutrition 

CSTL supports the food industry 

by providing reference measurements and reference standards for 

quality assurance and to help 

ensure compliance with the 

nutritional labeling regulations. 

CSTL provides robust metrological 

traceability for nutrients in food 

products, contaminants and adulterants in food products, chemical composition and contaminants 

in dietary supplements/nutraceuticals, and detection of genetic modifications in food products. 

Completion of Suite of SRMs to Support Nutritional Labeling Issues 

K.E. Sharpless, J.M. Brown Thomas, S.A. Margolis, B.C. Nelson, C.S. Phinney, J.R. Sieber, 

and L.J. Wood (839) 

CSTL’s Analytical Chemistry Division is working in several areas within the 

broad category of “food and nutrition” to facilitate compliance with nutritional 

and dietary supplement 

labeling laws, provide 

traceability for food exports 

needed for acceptance in 

many foreign markets, and 

improve the accuracy of 

information that is provided 

on product labels to assist 

consumers in making 

sound dietary choices. The 

composition of a food-matrix SRM should be 

matched closely to that of the test sample being 

analyzed; a fat-protein-carbohydrate food 

composition model developed by AOAC 

International is a useful way to classify the food matrix. NIST has been working with other federal 

agencies and the food industry over the past several years to provide a suite of SRMs within this 

model to address nutrition-labeling issues. 

Their positions in the AOAC triangle, along with 

those of the other food-matrix reference materials 

available from NIST, are shown. This particular 

aspect of NIST’s food and nutrition program can be 

expected to decrease. Focus will then shift to more 

specialized analytes (e.g., individual amino acids, 

allergens, mycotoxins) in foods, as well as to 

dietary supplements. 

CSTL/NIST develops reference 

materials and analytical methods to: 

• underpin measurements made for 

compliance with nutritional and 

dietary supplement labeling laws 

• provide traceability for food exports 

needed for acceptance in many 

foreign markets 

• improve the accuracy of information 

that is provided on product labels to 

assist consumers in making sound 

dietary choices.

NEW SRMS COMPLETE AOAC TRIANGLE

SRM 1946 Lake Superior Fish Tissue 

SRM 2384 Baking Chocolate 

SRM 2385 Spinach 

SRM 2387 Peanut Butter 

The U.S. processed food 

and beverage industry is a 

major participant in the 

global market. Almost half 

of the world’s top 50 food 

processing firms are 

headquartered in the U.S. 
68

Reference Methods and Materials for the Chemical Characterization of Dietary 

Supplements 

S.A. Wise, K.W. Phinney, L.C. Sander, and K.E. Sharpless (839) 

Taxonomically authentic botanical reference materials with assigned values for active and/or 

marker compounds are needed for quality assurance of analytical measurements associated with 

the manufacturing process and for the verification of 

manufacturers’ label claims for the dietary 

supplement industry. Assigned values for 

contaminants and adulterants are also necessary to 

address public health and safety concerns. In 2001, 

NIST, the Food and Drug Administration (FDA), and 

the National Institutes of Health’s Office of Dietary 

Supplements (NIH-ODS) initiated a multi-year 

program to develop 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. Ephedra-containing materials were identified as the 

highest priority by FDA and NIH-ODS, and we are currently in the process of producing a suite of 

five ephedra-related SRMs to address this need. We 

anticipate that the ephedra SRM suite will be 

completed by September 2003. 

It is necessary to provide SRMs and methods that 

address two needs in the dietary supplements area: 

safety and efficacy. Potential health risks may result 

from contamination (e.g., pesticides, heavy metals), 

adulteration (presence of unlabeled foreign materials 

including pharmaceuticals), or variability in product 

composition (e.g., changes in levels of active 

constituents). Secondly, product quality and 

consistency must be maintained, through verification 

of dietary supplement label claims. These needs 

can be addressed by the development of analytical 

methods and reference materials to support 

chemical measurements for dietary supplements. 

More than 50% of the U.S. 

population uses dietary 

supplements, accounting for 

roughly $10 billion in sales every 

year. A variety of botanicalcontaining products are marketed 

as dietary supplements; common 

examples include St. John’s wort, 

ephedra, and ginkgo. 

FDA and NIH-ODS identified ephedracontaining materials as the first 

priority for development of dietary 

supplement SRMs. A suite of 

materials is being produced, 

consisting of authentic ephedra plant 

material, two extracts of the plant 

material, and two finished products. 

These SRMs will be characterized for 

the ephedrine alkaloids, as well as for 

contaminants (Hg, Cd, Pb, As, and 

pesticides). 
69

Evaluation of Extraction Methodologies for Corn Kernel (Zea mays) DNA for Detection of 

Trace Amounts of Biotechnology-Derived DNA 

M.J. Holden and J.R. Blasic Jr. (831) 

Testing for the identification of biotechnologyderived plant material is currently required for 

compliance with labeling regulations implemented 

by countries other than the United States. 

European Union regulations mandate labeling of 

plant material and food as “genetically modified” if 

the material is present at a level of 1 % by weight. 

As a result, testing is important for trade in U.S. 

agricultural products, especially for the export of 

two commodity crops, soybeans and corn. Testing 

involves detection of the specific DNA that defines 

the modification in the plant genome. The “needle 

in a haystack” is relevant imagery. The polymerase 

chain reaction, or PCR, is a powerful tool to detect 

the target DNA or “needle” by synthesizing many 

copies of the DNA. Specific and consistent 

detection is required for accurate identification of a 

particular genetic modification in a plant sample, 

especially when the modification is present in trace 

amounts. 

The first important requirement for success with 

PCR is the isolation of DNA of high quality and in 

sufficient quantity to be representative of the sample of plant material. In this study, four 

investigators in two laboratories (NIST and USDA) evaluated six different methodologies. The 

plant material from which DNA was isolated consisted of ground corn kernels that contained 0.1 

% by weight of biotechnology-derived corn of a specific type 

known as StarLink. DNA was extracted using five 

commercial kits plus a published protocol, a variation of a 

method known as CTAB / NaCl. The particle size of the 

corn flour used for the DNA isolations was evaluated along 

with the quality and efficiency of extraction of the DNA from 

ground corn. The isolated DNA was then tested in three 

different PCR assays to ascertain the amplifiability of the 

target DNA. The PCR assays resulted in some “false 

negative” results, that were most likely due to inhibitors of 

the PCR process that co-purified with the DNA. 

The study highlights several aspects that should be considered 

when choosing a method for DNA extraction. The sample from 

which DNA is extracted must be representative of the material 

from which it is taken. Important parameters in this case include 

sufficient mass and fineness of the grinding plus the extraction 

efficiency of the method. Many individual samples must be 

tested for quality and amplifiability to evaluate a particular 

method. While several, but not all, methods performed 

reasonably well, the study showed that additional purification 

steps are necessary to accurately and consistently detect trace 

amounts of target DNA. We demonstrated that a commonly 

CSTL researchers investigate 

the quality of genomic DNA, 

isolated using protocols based 

on some common principles 

(binding to matrix or selective 

precipitation), as material for 

testing for the presence of 

biotechnology-derived corn 

The results of this study are 

of practical importance for 

testing laboratories of all 

kinds that have a mission to 

identify genetic components 

of grain. Accurate 

identification is critical to 

U.S. international trade in 

commodity crops. 
70

Soybeans 

used criterion for verifying the quality of isolated genomic DNA for PCR, amplification of an 

endogenous gene (present in all corn DNA), is not sufficient and can lead to false negative 

results. Inhibitors of the PCR process, that is corn kernel 

components that are isolated along with the genomic DNA, 

are likely culprits in false negative results. Our 

investigations show that acidic polysaccharides act to 

inhibit the PCR reaction, while starch, a major 

polysaccharide component of corn kernels, does not. The 

identification of specific inhibitors and their mode of 

interference in DNA amplification process should be 

addressed. The effect of inhibitors on the quantitation of 

DNA and the efficacy of various processes for removal of 

inhibitors will be investigated. 

Reference Methods and Standards for Transgenic Proteins in Biotech Foods

D.M. Bunk (839) 

There are currently no reference methods for the detection of transgenic proteins in biotech 

foods. Therefore the reference materials that are now being used have not been validated by 

methods other than field methods (i.e., immunoassays). 

Because foreign trade barriers exist for grains based on the 

amount of biotech grains present, improving the accuracy of 

field methods for transgenic protein detection could have a 

substantial impact on 

international grain sales. 

The immunoassays and 

lateral flow test strips that 

are currently used in the 

field method for the measurement of transgenic protein in 

biotechnology food products rely on antibodies to detect the 

protein expressed as the result of the specific genetic 

modification. The use of antibodies imparts high selectivity 

and sensitivity to protein-based measurements, in addition to 

making them rapid, robust, and relatively low-cost. However, 

the highly selective binding of the antibody to the protein 

antigen can also be problematic. If structural changes in the 

antigen occur, such as post-translational modifications or 

degradation occurring during sample preparation, 

antibody/antigen binding can be diminished, affecting assay 

results. Measurement procedures that do not rely upon 

antibody selectivity, must be developed to validate field 

methods and for the eventual certification of reference 

materials. As a 

starting point to the 

development of a reference method, a proteomic 

approach was explored for these measurements. 

Without the use of antibodies, the transgenic protein in 

glyphosate-tolerant soy and corn, CP4 EPSP 

synthase, was detected in crude protein extracts by 

mass spectrometry after tryptic digestion of the protein 

extract and multi-dimensional chromatographic 

separations. 

Similar studies on DNA 

extraction should be conducted 

on relevant plant materials with 

different compositional analysis. 

It would be of value because 

different problems would be 

encountered. An example is 

soybean, which contains 

significant lipid content, not 

found in corn. 

CSTL develops analytical 

methods based on 

chromatography and mass 

spectrometry to detect 

transgenic proteins without 

the use of antibodies and 

immunoassays. 

The results of this work demonstrate 

that transgenic proteins could be 

detected without the use of antibodies 

and immunoassays, and provide the 

groundwork for the development of 

quantitative reference methods based 

on chromatography and MS for 

transgenic proteins in biotech foods.
71

8. Forensics and Homeland Security 

CSTL has provided DNA standards for human 

identification since 1992; however, over the 

past year the effort has focused on new 

methods of analysis to respond to the need to 

analyze extremely degraded samples. Much of 

this was stimulated by the need to identify 

victims from the World Trade Center collapse. CSTL has also had a 

long history of assessing standards needs and developing new 

measurement technologies for crime scene investigations in which 

handguns or explosives are used. Reference standards are used by 

the forensic community both for quality assurance and to provide 

legally defensible analyses. 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 represent a major new area of 

expansion for CSTL. 

Y Chromosome Assays and Standards Support Commercial Technologies 

J.M. Butler, R. Schoske, P.M. Vallone, J.W. Redman, and M.C. Kline (831) 

Y chromosome short tandem repeat (STR) and single nucleotide polymorphism (SNP) markers 

have a number of applications in human identity testing including typing the perpetrator of sexual 

assault cases without differential 

extraction and tracing paternal lineages 

for missing persons investigations. In 

order for Y STR systems to become more 

widely accepted within the forensic DNA 

typing community, robust multiplex assays 

are required that will permit collection of 

information from many sites along the Y 

chromosome from a very minute amount 

of template DNA. We have focused on the design and development of new Y STR multiplexes as 

well as evaluating various markers in the same reference set of DNA samples. 

Commercial Testing Kits: Promega Corporation is developing a 12plex Y STR 

kit that is a subset of the markers contained within the published 

NIST Y STR 20plex. Promega is using many of our primers and 

building upon the information gained in our multiplex 

development. Orchid Cellmark has adopted the NIST Y STR 

10plex and completed population studies and forensic validation with this 

multiplex assay. Orchid plans to market their kit to members of the forensic DNA 

typing community. Both companies 

are looking to NIST SRM 2395 

Human Y Chromosome Standard to 

define their allele nomenclatures as 

is ReliaGene Technologies Inc. that 

currently has the only commercial Y STR kits 

available. In addition, genetic genealogy 

companies such as Relative Genetics and 

FamilyTree DNA are using our 20plex or 

portions of it in their genetic data collection. 

For Y SNP assays, we have beta-tested the 

New Y chromosome assays will permit more 

rapid collection of information on the 

variability of Y STR and Y SNP markers in 

human populations. A Standard Reference 

Material (SRM 2395) is under development to 

aid in reliability of measuring Y chromosome 

DNA typing results. 

NIST is a member of the 

FBI’s Scientific Working 

Group on DNA Analysis 

Methods Y chromosome 

subcommittee and is helping 

to define the core set of Y 

STR and Y SNP markers that 

will be used in future 

national and international 

DNA databases. 
72

Signet Y SNP Identification System from Marligen Biosciences, Inc. 

and provided them with data on the usefulness of their DNA 

markers.

The new NIST SRM 2395 Y Chromosome, 

contains extracted DNA from five unrelated and 

anonymous male individuals with sequence 

information from over 20 different and 

commonly used Y STR markers. 

Developing and Evaluating New Forensic Tests for Probing the Mitochondrial Genome 

P.M. Vallone, M.C. Kline, and J.M. Butler (831) 

The mitochondrial genome consists of ≈16,569 base pairs. 

Mitochondrial DNA is maternally inherited and can be 

employed in forensic investigations. The mitochondrial 

genome is much smaller than the nuclear human genome 

(3 billion base pairs). However, thousands (1000 to 5000) 

of copies of the mitochondrial genome are present per cell 

versus 2 copies of nuclear DNA. Due to the large number 

of mitochondrial genomes per cell it is often easier to type 

samples exposed to harsh environmental conditions i.e. 

when it is impossible to type the nuclear DNA. 

A stretch of ≈ 1100 base pairs in the control region of the mitochondrial genome is commonly 

analyzed for forensic purposes. The control region is highly polymorphic and is typically analyzed 

by DNA sequencing methods. Recently a linear array system from Roche 

Molecular Systems has been developed for rapid determination of common 

polymorphisms in the control region 

(specifically HV1 and HV2). Our 

laboratory was asked to participate in 

the beta testing of these “mito-strips”. 

These linear arrays offer forensic DNA 

laboratories a rapid test for screening 

samples and excluding ones that do not 

match prior to the labor-intensive effort 

of full sequencing. 

Separation and sequencing of alleles for the 

Y STR marker DYS385, which is duplicated on 

the Y chromosome gives rise to two bands 

that must be separated prior to sequencing. 

The number of GAAA repeats observed in the 

prototype SRM 2395 samples confirms allele 

calls by the commercial kit tested. 

CSTL researchers develop novel 

multiplex SNP assays for 

increasing the power of 

mitochondrial DNA for human 

identifications purposes; and 

evaluating “mito-strip” linear 

arrays from Roche Molecular 

Systems for rapid determination of

HV1 and HV2 haplogroups. 

This work serves to enhance 

the capability/capacity of 

mitochondrial DNA in human 

identification testing. In many 

cases (such as mass 

disasters) mitochondrial DNA 

is the only means to identify 

an individual.
73

In addition to evaluating control region screening 

assays, we are developing new SNP assays that 

can probe sequence polymorphisms in the 

remainder of the mitochondrial genome (the coding 

region). This coding region test will allow for 

discrimination between individuals who possess 

common (and undistinguishable) control region 

sequences. Currently eleven polymorphic sites are 

probed in our coding region multiplex test. The 

coding mitochondrial DNA work is being performed 

in collaboration with the Armed Forces DNA 

Identification Laboratory in Rockville, MD. 

Providing Assistance in the DNA Identifications of World Trade Center Disaster Victims 

J.M. Butler, P.M. Vallone, G. Spangler, and M.C. Kline (831) 

The high degree of fragmentation of the individuals killed in the 

World Trade Center (WTC) disaster of September 11, 2001 has 

meant that forensic DNA typing is the primary means employed to 

identify the victims’ remains. The bones and tissue samples 

collected from the WTC site are highly degraded due to the 

severe environmental conditions experienced during the collapse 

of the twin towers and the fires that raged for months afterwards. 

The degraded samples have posed a challenge to the current 

state of the art in forensic DNA typing technology. Only about 

50% of the specimens are yielding results with standard short 

tandem repeat (STR) 

DNA testing methods. 

NIST is assisting the ongoing identification efforts in 

several different ways. NIST has developed miniSTR 

assays that amplify smaller portions of the polymorphic 

sites used in standard forensic DNA tests. The smaller 

PCR product sizes potentially make these assays more 

sensitive and more capable of recovering typing 

information from degraded DNA specimens. 

Ten mitochondrial polymorphisms are 

detected in a single test using a 

SNaPshot assay developed at NIST 

CSTL Researcher John Butler serves 

on the WTC Kinship and Data 

Analysis Panel (KADAP) organized by 

the National Institute of Justice. The 

KADAP panel is composed of 25 

experts from around the country to 

evaluate new technologies and 

genetic analysis approaches that 

might be used on the WTC samples. 
74

Results obtained from a multiplex SNP detection assay developed at NIST to evaluate the 

allele calls produced by the commercial SNP markers. A paternal grandfather (PGF), paternal 

grandmother (PGM), and father of a family reference set were used to demonstrate expected 

genetic inheritance with these 6 SNP markers. The father is heterozygous at all 6 loci shown 

here. SNPs are being considered for use in WTC samples due to their ability to be amplified 

from degraded DNA as a very small PCR product (60 bp to 80 bp). 

Our original miniSTR assays were 

developed at the request of Dr. Robert 

Shaler, the head of the head of Forensic 

Biology section at the Office of the Chief 

Medical Examiner of New York (NYCOCME) who is leading the WTC DNA 

identification effort. A miniSTR assay 

dubbed “Big Mini” was provided to the 

NYC-OCME in January 

2002. Based on the 

groundwork laid with 

our miniSTRs, the Bode 

Technology Group has developed 

commercial miniSTR assays that are 

being used to enhance the recovery of 

STR genotypes from WTC samples. 

Information from over 20,000 bones and 

tissue fragments is being analyzed to help 

make the DNA identifications of the WTC 

victims. 

NIST has also assisted in the validation of new 

genetic markers that are being evaluated for their 

usefulness in human identification. Orchid Cellmark 

has developed a panel of 70 autosomal SNP 

markers with PCR product sizes in the range of 60 

bp to 80 bp that can produce results on degraded 

DNA samples. In a matter of a few weeks, the NIST 

team developed new assays with the commercial 

markers. Over 500 SNP calls were evaluated and 

found to be fully concordant between the two 

different assays. 

Comparison of allele calls at the STR marker 

D16S539. In both cases, a 9-11 genotype was 

obtained demonstrating concordance between 

the two assays. The new miniSTR test reduces 

the overall PCR product size by >150 bp. 
75

Standardization of Sample Preparation Techniques Used in Analyzing Microorganisms by 

MALDI-TOF Mass Spectrometry 

C. Nelson, B.J. Porter, M.J. Welch, B.C. Nelson, E. White V (839); and W. G. Mallard (838) 

The rapid and reliable detection and identification of microorganisms has 

become a crucial issue for homeland security. Current CSTL efforts, 

supported by funding from the Defense Threat Reduction Agency (DTRA), 

are focused on standardization of methods for identification of 

microorganisms using Matrix-Assisted Laser Desorption/Ionization Time-ofFlight Mass Spectrometry (MALDI-TOF MS). Approaches for sample 

preparation and data interpretation of spectra obtained using this technique 

must be critically evaluated to provide further database input and search 

algorithm development. Essentially, proteins 

expressed by bacteria, both intracellularly and on 

the bacteria surface, can produce characteristic 

mass fingerprints useful in distinguishing between 

organisms, even at the strain level. Slight variations 

in sample handling have been observed to 

drastically affect the resulting mass spectra for both 

Gram negative and Gram positive bacteria, hence the need for determining which techniques 

provide the best and most reproducible results. Interlaboratory 

comparisons of mass spectral data obtained on different 

instruments showed that common ions could be reproducibly 

observed when a standard protocol was followed. Standardization 

of bacterial sample preparation and analysis by MALDI-TOF MS, 

along with algorithmic development for library searches, will not 

only aid in the identification of bioterrorism agents for homeland 

security, but has the potential to impact other areas (e.g., biological 

weapons treaty violations, environmental, biomedical research, 

clinical, food industry). 

Matrix selection was observed to have the greatest impact 

on MALDI-TOF mass spectral patterns of the lyophilized 

Escherichia coli (Gram negative) and Bacillus subtilis (Gram 

positive) samples tested. Disruption of lyophilized E. coli

cells by ultrasonication or extraction of proteins with certain 

solvents produced mass spectra that were very similar to 

whole cell analysis, with the exception of several higher 

molecular weight ions between 20 kDa to 40 kDa present in 

the former. Data collected on different samples of E. coli

indicated the presence of common biomarkers among the 

strains tested, as well as markers that may be specific to 

each strain. Comparisons of the spectral patterns of two 

preparations of a single strain of B. subtilis from two different 

sources indicated the presence of many common ions, 

which may be used to fingerprint the organism, regardless of 

its origin. An interlaboratory comparison study provided 

evidence to support the feasibility of MALDI-TOF MS as a 

rapid technique for bacterial identification in field applications 

when different instruments are used. Although different 

laboratory conditions and instruments were used, common 

ions were observed reproducibly across the laboratories when a standard protocol was followed. 

CSTL researchers standardize 

bacterial sample preparation and 

analysis by MALDI-TOF MS to 

facilitate algorithmic development of

library searches to assist in the 

identification of microorganisms.

Bacillus subtilis

Escherichia coli
76

2000 5200 8400 11600 14800 18000 Mass (m/z)

0

20

40

60

80

100

% Intensity

MBT

0

20

40

60

80

100

% Intensity

alpha-CHCA

0

20

40

60

80

100

% Intensity

Ferulic Acid

0

20

40

60

80

100

% Intensity

Sinapinic Acid

MALDI-TOF mass spectra of whole cell E. coli

ATCC 9637 (strain W) using four different 

matrixes: sinapinic acid, ferulic acid, alphacyano-4-hydroxycinnamic acid (alpha-CHCA), 

2-mercaptobenzothiazole (MBT). 

With the support of the Defense Threat Reduction Agency (DTRA) Chemical/Biological Arms 

Control Program, this project is expected to continue for the next 2 to 3 years. NIST will continue 

to examine the various experimental 

parameters affecting the spectra of test 

microorganisms, e.g., matrix, wavelength 

and intensity of the laser, and the nature of 

the sample (ionic strength, growth media, 

etc). Variability due to small changes in 

the protocol will be examined to determine 

how exacting the specifications of the 

standard operating procedure must be. A 

specific set of standard conditions that 

produce reproducible spectra under the 

widest set of normal instrument variations 

will be determined. Standard operating 

procedures for the production of spectra 

will be developed and tested in 

interlaboratory round robin studies with the 

goal to create a library of MALDI-TOF MS 

for a wide variety of microorganisms. 

Precision Isotopic Measurements with the NIST High Performance Secondary Ion Mass 

Spectrometry Facility 

A.J. Fahey (837) 

The specific isotopic ratios of an element in a 

sample can, in some cases, be used to determine 

the history of a sample, its age, or its place of origin. 

In order for isotopic ratio data to be useful it often 

must be determined to a precision and accuracy of 

less than 1 part per thousand. To this end we have 

characterized the new NIST ims-1270 secondary ion 

mass spectrometer and its subsystems for highprecision measurements of isotopic ratios. 

Samples of known chemical and isotopic 

composition are available in the form of U3O8 as 

Standard Reference Materials. These SRMs are 

unique in that they span more than 4 decades in 

their major isotope ratio. In order to characterize the 

ability of the ims-1270 at NIST to measure uranium 

isotopic compositions precisely and accurately, a 

selection of uranium SRMs were measured where 

the abundance ratio 235U/238U ranged from ≈190 

(highly enriched) to ≈ 0.7 (natural). Combined with 

the minor isotopes of uranium, this scale of variation 

Deviation in 235U/238U isotope ratios 

from certified values. 

Certificate Value of 234U/238U

10-5 10-4 10-3 10-2 10-1 100 101

Fractional Deviation of 234U/238U -0.03

-0.02

-0.01

0.00

0.01

0.02

0.03

U750 U900 U970 U500

U350

U350 U100 U100

NBL010

U030a U950

U950

Certificate Value of 235U/238U

10-3 10-2 10-1 100 101 102 103

Fractional Deviation of 235U/238U -0.008

-0.006

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

U970

U970

U970

U900 U750 U500

U350U500

U350

U100

U100

NBL010

U030a

U030a

U950

U950

Certificate Value of 236U/238U

10-6 10-5 10-4 10-3 10-2 10-1 100

Fractional Deviation of 236U/238U

-0.2

-0.1

0.0

0.1

0.2

0.3

U970

U500 U750 U900 U970 U350 U100 U350 NBL010

U030a

U030a

Mass Fractionation Correction

of 0.332 %/amu Applied to All Data.

CSTL researchers measure isotopic 

ratios with high precision and 

accuracy in order to characterize 

microscopic quantities of materials. 
77

allows us to nearly span the dynamic range of the pulse-counting detection system. In addition, it 

allows us to test the reproducibility of the mass fractionation for isotopic ratio measurements on 

particles. 

The figure shows the deviations of each of the measured isotopic ratios for the available uranium 

SRM materials as a function of the certificate values for each SRM. The data shown was taken 

over a period of four months from May to September of 2002. A constant mass fractionation was 

applied to all of the data. Nearly all of the data points lie within one standard error of the mean for 

each of the isotopes and SRMs. There are two exceptions to this and the reasons for each are 

well understood. There is a trend in the 235U/238U values that is apparently dependent on the 

isotopic ratio. This is due to the fact that there is a non-linearity in the response of the electron 

multiplier detector used to count the ions. This effect has 

been documented by others including the group at New 

Brunswick labs that currently curates these SRMs. The 

magnitude of this non-linearity is very small, 

approximately 4 parts per thousand spanning more than 

4 orders of magnitude (that is, <1 μg/g). The other 

significant deviation from the certified value lies in the 

measurement of 236U for the SRM U970. This isotope of 

uranium, 236U, is the least abundant isotope in all of the 

uranium SRMs, and in U970 it is the most difficult to 

measure because of an unresolvable interference due to 

235UH. Although the hydride interference correction can 

be made reliably for most of the SRMs the U970 is 

unusual because of its very high abundance of 235U, generating a large 

235UH signal, and its low abundance of 238U (the 238UH signal is used to 

correct for the interference of 235UH at the mass of 236U). However, our 

data indicate that by using the “cold trap” on the ims-1270 sample chamber, 

we can reduce the hydride intereference enough to make an accurate 

correction. 

Microfluidic Whole-Cell Biosensors for Early Detection of Toxins in Water Streams 

L.E. Locascio, E.A. Waddell, J.C. Travis (839); and A. Suggs, B.J. Love, and N.G. Love 

(Virginia Polytechnic and State Univ.) 

This collaborative research effort between the NIST and Virginia Polytechnic and State University 

involves the development of a cell-based microfluidic biosensor that will be used to detect the 

presence of electrophilic toxins in water samples. Electrophilic toxins flowing from streams into 

wastewater treatment plants can have a profound 

detrimental effect on treatment processes by 

destroying the bioreactor that is at the heart of the 

plant. Based on earlier work by Love’s group, it 

was hypothesized that the presence of electrophilic 

toxins could be monitored by measuring the 

potassium efflux from whole cells exposed to this 

class of toxins. The project involves the immobilization of whole cells, Escherichia coli K-12, 

inside microfluidic channels and measurement of cellular potassium efflux in a microfluidic system 

using a potassium-sensitive optical sensor embedded in the device. Prototype devices will be 

evaluated using samples spiked with a range of electrophilic toxic chemicals. Ultimately, the 

device is targeted for the detection of toxic chemicals in wastewater streams, but may also be 

used to detect toxins in drinking water supplies. 

Different classes of chemicals feeding into water treatment plants have been known to cause 

bioreactor degradation leading to system processing failures. A recent example of this occurred 

The measurements reported here 

show that the new NIST ims-1270 is 

capable of high-precision and 

accurate measurement of U3O8

particles. The mass fractionation is 

stable and reproducible and 

corrections can be made reliably for 

hydride interferences. This will aid 

in our support of the International 

Atomic Energy Agency (IAEA) for 

monitoring signatories of the 

Nuclear Non-Proliferation Treaty. 

Collaborative research efforts lead 

to the development of a sensitive 

and reliable biosensor to detect the 

presence of electrophilic toxins in 

wastewater streams. 
78

L) Fluorescence image of optode 

membrane with no K+

 in solution. 

R) Image of membrane with 50 

mM K+

 in solution 

earlier this year at the Hagerstown water treatment facility upstream of Washington, DC where 

millions of gallons of raw sewage were dumped into the Potomac River as a result of chemically 

induced bioreactor failure. 

Electrophilic toxins are one class of chemicals that 

can impact bioreactor function and cause a 

chemically induced failure. In this collaborative effort, 

we have designed a cell-based microfluidic biosensor 

to detect the presence of electrophilic toxins in water 

streams entering wastewater treatment plants in an 

effort to prevent failure of the bioreactor. The 

approach is to immobilize bacterial cells in a 

microfluidic system and monitor cell response, in 

particular potassium efflux, as the water flows 

through a bed of immobilized cells. In essence, we 

have created a micro-bioreactor. The behavior of our micro-bioreactor is then used to predict the 

behavior of the process bioreactor when exposed to the same water supply. A negative response 

in the microsystem can be used to decide when to divert part of the incoming stream to prevent 

process failure. 

The microfluidic system that 

we have designed for this work 

has two main components: (1) 

a bed of immobilized E. coli

cells creating the microbioreactor; and (2) a reversible 

potassium sensor. The cells 

have been immobilized by 

adsorption directly onto the 

polymer microchannel surface. 

It was determined that there 

was greater binding and 

greater cell viability if the cells 

were grown in batch to the late 

log phase and then seeded in 

the microchannel. Cells that were seeded for 4 h to 8 h exhibited a stable biofilm. Overgrowth of 

the cells in the micro-bioreactor is a concern, however, so we are currently investigating other 

mechanisms for immobilizing cells to discourage fast growth that could lead to channel occlusion. 

The reversible potassium sensor was a polymer film that 

contained a potassium sensitive ionophore and a 

chromoionophore that responded to a local pH change 

with a change in fluorescence intensity. Fluorescence 

images of the film before and after exposure to potassium 

ions are shown in the figure. The optode was placed 

downstream of the immobilized cells and was used to 

monitor the release of potassium ions from inside the cells 

in the presence of an electrophilic toxin. Optimization of 

the optode film and incorporation of appropriate control 

films are being studied to improve the performance of the 

device. The goal is to produce a simple and reliable 

system that can be placed in the field and used continuously. 

The focus of this collaborative work is 

the in-situ monitoring of environmental 

toxins that could seriously impact the 

efficacy of bioreactors used in 

wastewater treatment processes. A 

reliable early warning system such as 

the one here can have a profound 

environmental and financial impact by 

detecting the problem before a process 

upset occurs. 

Cell-lined 

channels 

Cell-free 

channels 

Cell-free 

channels 

Optode:

K+

 specific 

chromoionophore 

film

Cell response to toxin measured by K+ 

specific optode 

Spectrometer 

Water 

stream 

Schematic of cell-based microfluidic biosensor 

system 
79

Chemical Warfare Agent Detection with Microsensor Arrays 

C.J. Taylor, R.E. Cavicchi, D.C. Meier, and S. Semancik (836) 

MEMS-based (Microelectromechanical System) microsensors were used to detect chemical 

warfare agents in a project supported by the Defense Threat Reduction Agency (DTRA). The 

microsensors are based on surface-micromachined 100 μm x 100 μm platforms called 

microhotplates. The agents are detected by 

measuring conductance changes in metal oxide 

thin films deposited on the microhotplates; the 

sensor response is correlated with the identity 

and concentration of the chemical agent 

present. 

Studies done at NIST and a surety laboratory 

have established that conductometric 

microsensors based on oxide sensing films and 

temperature-controllable MEMS platforms can 

detect a variety of chemical warfare agents. 

These types of microsensors may some day be 

used to warn military personnel and civilians of releases of chemical 

warfare agents. As part of the work, the sensing performance of 4-

element microsensor arrays to the simulant molecules of chloroethyl 

ethyl sulfide (CEES) - a sulfur mustard simulant, and diisopropyl 

fluorophosphates (DFP) - a nerve agent simulant, was also evaluated. 

The simulant experiments were performed to develop sensing materials 

and operating schemes suited to the classes of compounds used in 

chemical warfare. The first phase of testing on three agents, sulfur 

mustard gas (HD) and the nerve agents tabun (GA) and sarin (GB) was 

completed in experiments conducted at the Edgewood Chemical 

Biological Center (ECBC). 

Microsensor response signals were generated as changes in 

resistance (conductance) of thin SnO2 and TiO2 films as they 

were exposed to the agents. The nanocrystalline sensing films 

were fabricated on microhotplate platforms by chemical vapor 

deposition, and operated between 20 °C and 480 °C in both 

fixed- and variable-temperature modes. These tests 

demonstrated that the devices can detect the agents in the 

0.01 μg/g (ppm) to 0.3 μg/g (ppm) range with adequate signalto-noise. Good stability and reproducibility were observed 

over 14-hour continuous monitoring runs. Since “LC50” 

concentration levels for 10-minute exposures are ≈1 μg/g (ppm) for the agents tested, it is clear 

that the microsensors are sensitive enough for a practical monitoring technology. 

Response to GB (375 °C) 

 #1 SnO2 #2 

 #1 TiO2 #2 600 nm 

Nano-crystalline tin and titanium oxide sensing films on in a 4-element 

microhotplate array with conductance response 

Microsensors are tuned for specific 

agents by incorporating metal oxide films 

of different composition into arrays of 

sensors, and by selecting different fixed 

and time-varying temperature programs. 

Studies were first conducted on simulant 

agents at NIST to develop appropriate 

device prototypes for testing the 

technology on actual sulfur mustard and 

nerve agents at a surety laboratory.

The NIST microsensor 

technology offers other 

desirable features for agent 

detection including low power 

consumption, low cost, ease 

of including redundant 

devices, and ability to readily 

integrate on-board electronics 

for signal conditioning. 

ECBC 
80

Verification of Calibration Procedure for Testing Military Gas Masks 

R.A. Fletcher, R.L. King, J.A. Small (837); M.R. Winchester (839); J.C. Yang, and G.W. 

Mulholland (BFRL) 

Military personnel wear gas masks for protection 

against a chemical and biological assault. The 

recognized potential global threat from chemical 

and biological agents demands that effective 

defense measures be taken. An important part of 

this defense strategy is the testing and 

verification that assures secure gas mask fits. 

Currently, the Army determines the gas mask fit 

quality by measurement and comparison of the 

ambient aerosol concentration outside of the 

fitted masks to the aerosol concentration inside. 

The particles can identify both leaks in the mask 

and inefficiencies in the filters. This technology is 

believed to provide a complete diagnostic of the 

integrity of the mask, the filter, and the fit on the 

individual. 

The Army, working with TSI, 

Inc., an aerosol instrument 

manufacturer, has developed the 

M41 Protective Assessment Test 

Systems (PATS) to test and 

verify the goodness of fit for the 

gas mask on the individual 

person that is issued the device. 

The M41s are tested and verified by the U.S. Army Test, Measurement, and 

Diagnostic Group using a fit-test calibration stand (test stand). The components 

of the test stand are an aerosol generator, aerosol electrical charger, a mobility classifier to 

produce monodisperse 80 nm diameter aerosol, an aerosol dilution system and two condensation 

particle counters (CPCs). The test aerosol concentration range is 100 particles/cm3 to10,000 

particles/cm3

. The CPCs serve as the calibrants in the test stand for the M41s. There are no 

aerosol concentration standards for the CPCs nor are there certified reference materials available 

for aerosol concentration standardization. The measurement process currently has no links or 

traceability to a standards granting organization. The U.S. Army Test, Measurement, and 

Diagnostic Equipment Activity (USATA) requires the development of an aerosol concentration 

standard method, traceable to NIST. 

CSTL has provided a NIST traceable calibration for the 

aerosol electrometer. This allows us to verify the 

voltage response for known femtoampere current 

inputs. We have finished the calculations to simulate 

particle loss in the CPC. Electron microscopy has 

been completed on quantitatively collected 80 nm 

particles over a concentration range of approximately 

200 particles/cm3

 to 10,000 particles/cm3. This 

verification and possible development of a standard method would enable the accurate calibration 

of Condensation Particle Counters that are currently used to calibrate M41 Protective 

Assessment Test Systems (PATS) for the Army. 

With present technology and the 

transient nature of aerosols, a 

certified reference material for aerosol

concentration has not been feasible. 

Therefore, NIST verified the accuracy 

of the calibrant currently used in the 

field by comparing aerosol 

electrometer measurements with 

those obtained with NIST’s primary 

particle counting approach using 

electron microscopy. 

This work is also directly relevant to 

homeland security as the procedures 

developed here are applicable for 

verification of instruments used to fit 

test gas masks and protective suits 

used by first responders and 

emergency response personnel. 
81

Characterization of Trace Explosive Detection Equipment for Airport Security Applications 

J.G. Gillen, R.A. Fletcher, S.A. Wight, S.V. Roberson, E.S. Etz, A.P. Lindstrom, C.J Zeissler, 

and J.R. Verkouteren (837); B.A. Benner (839); and R. Lareau (Transportation Security 

Administration) 

CSTL is responding to the growing metrology requirements for trace explosive 

screening by leveraging existing expertise in particle analysis, analytical chemistry 

and chemical microscopy to build a measurement infrastructure for the continued 

development, characterization, calibration and standardization of trace explosive 

detection devices. 

Since the low volatility of most high 

explosives makes direct analysis of vapors 

impractical, the majority of explosive detection 

systems rely on the collection of micrometersized explosive particles collected on swipe 

samples (tabletop units commonly used at 

airport security checkpoints) or from airborne 

particle collection (next generation walkthrough portal detection systems). Collected 

explosive particles are thermally desorbed 

from the collection medium (cloth or steel mesh) into an ion mobility spectrometer (IMS) for 

identification. The effective collection and subsequent thermal desorption of explosive particles 

into the IMS is the critical front-end process for the successful and reproducible detection of 

explosives. In FY02, we initiated a new 3-year research program (through NIST’s Advanced 

Technology Program Funding) to study this process in detail. A complete understanding of the 

operational parameters and limitations of trace explosive instruments is required for their effective 

and reliable operation. 

A number of experiments were initiated to evaluate both swipe 

and portal-based explosive detection systems. A priority has 

been to study the factors involved in the release and effective 

collection of explosive particles in portal systems. One current 

study involves the use of fluorescence optical microscopy to 

study the release of monodisperse, fluorescently tagged 

microsphere particles from model surfaces under an impinging 

air jet (as used in airborne collection systems). Preliminary data 

suggests that almost no particles with diameters less than ≈ 6 

μm are liberated under these sampling conditions. This 

information constrains the size of particles likely to be sampled 

using airborne collection techniques and will aid in the design of 

next generation explosive detection systems. The particle size 

collection efficiency of the 

metal collection filters used in 

portal systems is also being 

evaluated in this work. Another 

issue addressed this year was determining how to visualize a 

few individual explosive particles surrounded by millions of 

ambient aerosols on mesh filter collectors. For this purpose, 

new approaches for chemically specific imaging of explosive 

molecules were developed using the NIST cluster secondary 

ion mass spectrometry technique. 

A 

Researchers in CSTL and the 

Transportation Security Administration's 

(TSA) Trace Explosive Detection Group 

have been working to build a chemical 

metrology program to support the 

widespread operational deployment and 

effective utilization of trace explosives 

detection devices throughout the U.S. 

Current aviation security 

legislation requires the 

unprecedented utilization of 

trace explosives detection 

systems for screening of 

baggage, cargo, and 

passengers at US airports in 

support of Homeland 

Security.
82

A second major thrust has been to study the 

temperature dependence of explosive particle 

vaporization. In these experiments, secondary ion 

mass spectrometry, Raman microscopy, and 

scanning electron microscopy have been used to 

study the temporal variations in chemical 

composition and morphology that occur during 

heating of individual explosive particles. The figures 

show infrared camera images of two types of 

collection media used in commercial explosive 

detectors. The CSTL infrared imaging capability is 

used to provide precise non-contact and spatially 

resolved temperature measurements of these 

surfaces. This information provides a unique 

method for relating the chemical degradation 

information obtained using our microanalytical techniques to the actual temperatures achieved 

during the sampling process. In the next fiscal year, the scope of the work begun this year will be 

expanded to include a variety of additional studies. A robust examination of particle generation 

and collection will be conducted as well as a detailed evaluation of the thermal degradation of 

various explosives. New analytical capabilities in 

ion mobility spectrometry, gas chromatography, 

mass spectrometry, and optical and infrared 

microscopy are being added to support this effort. 

Autoradiography Measurements to Quantify Activity of Co-60 Particles

C.J. Zeissler (837); and R.M. Lindstrom (839) 

The physical nature of particulate matter presents special measurement challenges for forensic 

analysis of dirty bomb debris. For instance, conventional biological standards cannot be utilized. 

Standards for biology consist of a limited list of radiotracers in thick, tissue slices or gene 

separation gels. Particles, however, act as point 

sources, whose positional geometry can significantly 

influence the measurements. Another challenge is that 

the activity of particles in environmental dusts cannot be 

adjusted as one might in biological tracer studies, so that 

special treatment has to be given to signal-to-noise 

problems. Dirty bomb debris may include beta-emitting 

particles of Co-60, Cs-137, Sr-90, etc. Individual particle 

analysis enables the collection of a variety of discrete 

chemical, isotopic, and microstructural data on each 

particle in a population. These data may be used to deconvolve sources and histories. In 

contrast, information obtained from conventional methods such as gamma spectrometry or 

Infrared camera image of 

swipe collection cloth

Infrared camera image of 

stainless steel mesh used in 

portal explosive detection unit

Characterization of the performance 

characteristics of explosive detection 

systems is of significant importance for 

the TSA and law enforcement. Optimized 

performance will help to ensure the safety 

of our transportation system, minimize 

possible security threats and provide a 

more robust tool for state and local law 

enforcement applications. 

CSTL researchers are 

developing an autoradiography 

measurement approach to 

support forensic analysis on 

dirty bomb debris at a level 

that falls over one order of 

magnitude below conventional 

detection capabilities. 
83

radiochemistry provides a measurement averaged over 

the whole sample, and does not provide additional 

forensic information available in the microstructure or 

morphology of each particle. 

The digital autoradiography method has been shown to 

detect Co-60 nondestructively at a level at least one 

order of magnitude below low-background gamma 

spectrometry methods. Calibration down to 10 mBq 

has been achieved. Detection down to about 1 mBq or 

lower appears to be possible, but has not been verified 

at this stage of the project. Measurement uncertainty 

that is not due to counting statistics is 

approximately 10%. The difference in 

spatial resolution between two different 

detectors has been evaluated. This year we 

studied the fading characteristics of two 

phosphor detectors, measurement 

variability, phosphor calibration, spatial 

resolution, Co-60 detectability, and ease of 

adoption. In the future, we plan to produce 

working standards and a measurement 

protocol for potential use by national 

forensic authorities. 

A nondestructive, sensitive characterization 

method that preserves discrete particle 

information can have great forensic utility. 

Conventional methods should continue to 

be sufficient for conventional detection and 

characterization, but they have limited 

sensitivity and forensic value. With this new 

microcharacterization method, previously 

undetectable debris on dirty bombs or 

residues from alleged manufacturing 

locations could be used towards the 

identification of terrorist activities. Ultimately, such information can be used to prevent future 

manufacture or deployment of dirty bomb devices. 

20

25

30

35

40

45

50

55

60

0 60 120 180 240 300 360 420 480

fade time (min)

Signal Intensity (arb. Units)

Fading curves for two autoradiography 

phosphor detectors using a 3 Bq Co-60 

source. 

The purpose of this research into 

autoradiography measurements is 

to develop a quantitative, 

nondestructive and discrete 

measurement capability for 

particles that emit beta radiation at 

or below mBq levels. Single particle 

detection and characterization 

measurements of a particle 

population are applicable to 

forensic dirty bomb investigations. 
84

New Measurement Technologies and Standards to Aid in Crime Scene Investigations 

W.A. MacCrehan (839); M. Layman, J. Secl, and A. Dorsey (George Washington Univ.) 

Measurement technology and a quality assurance standard are being developed for the forensic 

investigation of crimes using guns and improvised explosive devices. A simple protocol was 

developed for the collection and analysis of the organic gunshot residue (OGSR) from hair using 

a fine-toothed comb. A series of weapon firing studies with both mannequin-supported wig hair 

and human shooters showed the hair 

combing approach to be highly successful. 

Currently, there are no quality assurance 

materials for the validation of analytical 

procedures for explosives determination or 

for the testing of laboratory proficiency. 

In an effort to support homeland security, we 

are developing a gunpowder reference 

material, RM 8107 Additives in Smokeless 

Powder. Gunpowder is the most common 

active ingredient in improvised explosive devices (IED) such as pipe bombs. Forensic 

investigators recover incompletely burned powder residues from crime scenes such as the 

Atlanta Olympic Park bombing (7/27/96), and these residues are analyzed for their additive 

composition as a means of categorizing the powder against known powder samples. A NIST 

smokeless powder reference material can be used by forensic laboratories to validate their 

analytical methodology and test the performance of laboratory personnel, assuring the accuracy 

of their smokeless powder compositional identifications. 

In our approach, the chemical composition of gunshot 

and explosives residues collected at crime scenes are 

determined by micellar capillary electrophoresis (CE). 

During the past year, we have made progress toward 

addressing two issues in successful gunpowder residue 

analysis: effective collection of gunshot residues and 

accurately determining the composition of explosives 

residues. A simple protocol for OGSR collection was 

developed using a fine-toothed comb (for the collection 

of fleas from pets) and direct analyte ultrasonic solvent 

extraction in a plastic zip-enclosure bag. This new 

OGSR analysis approach was evaluated in a series of 

weapon firing experiments using mannequin-supported 

human wig hair, which allowed us to determine 

successfulness in simulated shooter and victim 

scenarios. Four weapons were tested including a revolver, semiautomatic pistol, shotgun, and a 

carbine. In the majority of OGSR combing samples, the gunpowder additive nitroglycerin was 

successfully detected. The method also proved effective in detecting OGSR in human shooter 

testing. In the future, we intend to work toward the development of analytical measurements and 

standards for the analysis of the high explosives, such as plastic explosives, that are more 

commonly encountered in international terrorism investigations. 

CSTL researchers made compositional 

measurements of the organic additive 

content on the candidate smokeless 

powder Reference Material, RM 8107 

Additives in Smokeless Powder, which is 

being developed to help forensic 

investigators achieve better information 

from the collection of gunpowder residues 

at shooting and explosives crime scenes. 

OGSR

Image shows OGSR particles 

collected by combing a revolver 

shooter’s hair.
85

Reference Methods and Standards for Alcohol and Drugs of Abuse Testing 

M.M. Schantz, J.V. Goodpaster, L.T. Sniegoski, S. Tai, and M.J. Welch (839) 

Standard Reference Materials (SRMs) for use in the forensic sciences include: SRMs for blood 

alcohol determinations (SRM 1828a Ethanol-Water Solution), for measurements of drugs of 

abuse in urine (SRM 1507b THC-9-COOH; SRM 1508a Benzoylecgonine; SRM 2382 Morphine 

Glucuronide; SRM 8444 Cotinine; SRM 1511 

Benzoylecgonine, Morphine, Codeine, THC-9-

COOH, Phencyclidine; and SRM 2381 Morphine 

and Codeine), and in hair (SRM 2379 Cocaine, 

Benzoylecgonine, Cocaethylene, Amphetamine, 

Methamphetamine, and PCP; and SRM 2380 

Codeine, Morphine, Monoacetylmorphine, and 

THC). SRMs 2379 and 2380 for the 

determination of drugs of abuse in human hair 

were completed in FY02 and will provide this 

growing measurement technology with a 

mechanism for assuring the accuracy of the 

methods used to determine drugs of abuse in 

hair. 

Recently ACD, in conjunction with the NIST Office of Law Enforcement 

Standards (OLES), completed the evaluation of the specificity and sensitivity 

of spray reagents used by law enforcement officers in the field to test for the 

presence of drugs of abuse. Varying amounts of the target drugs were 

applied to the test strips supplied with the reagents and then treated with the 

sprays. Volunteers were asked to rate whether or not a positive response 

could be seen as a test of the sensitivity of the tests. Various substances that 

might be encountered by law enforcement officers were tested for 

interference with the drug tests. In general, the drug spray reagents 

performed in agreement with the manufacturer’s claim. In addition, three new SRMs are under 

development in response to feedback from the forensic community as to their most immediate 

needs for standards. The development of these new SRMs was initiated with support from OLES 

and include (1) an updated and redesigned SRM 1828b for blood- and breath-alcohol testing 

(SRM 1828a will be replaced by a set of nine solutions that will cover the concentration ranges of 

importance to both blood and breath alcohol testing), 

(2) an SRM designed for qualitative analysis of 

materials from an arson investigation, and (3) an 

SRM for drugs of abuse in human serum to 

complement the current SRMs for drugs of abuse in 

urine because forensic laboratories often receive 

blood instead of urine. 

Development of Graphical Tools for the Analysis of Data from Interlaboratory 

Comparisons 

D.L. Duewer (839) and M.C. Kline (831) 

For any experiment to have optimal utility, data and information must be transformed into 

knowledge and understanding. Forensic interlaboratory studies present a particular 

communications challenge since the audience potentially interested in the results (including the 

public, legal profession, participants, and agency sponsors) has inherently diverse backgrounds. 

Graphs that simply but accurately summarize results help meet this communications challenge. 

From anecdotal evidence, our graphical reports from earlier studies have led to improved forensic 

DNA measurement systems. 

Illegal use of controlled substances 

remains a serious problem in the U.S. 

Millions of tests are run annually to 

determine whether a person has 

ingested or possesses controlled 

substances. The incidence of false 

positives and false negatives from 

these tests impact public safety as 

well as the reputation of the 

individuals tested. It is therefore 

critical that such tests be traceable to 

accurate standards. 

With the completion of these SRMs, the 

law enforcement community will have 

several new tools to assure the 

accuracy of the methods used to detect 

drugs and substance abuse. 
86

-4 -3 -2 -1 0 1 2 3

Concordance, SD

Apparent Precision, SD

This "target" plot displays DNA concentration 

measurement comparabilities for the MSS3 

participants.

The MSS3 was initiated in late 2000, data 

were accepted until October 2001, and data 

entry and validation was completed as of 

June 2002. This interlaboratory challenge 

study was designed to help the forensic 

human identity community make more 

effective use of their limited resources by 

illuminating the relationships between their 

measurement of the concentration of DNA in 

extracted samples and the quality of their 

multiplex STR “fingerprints”. Analysts from seventy-four forensic laboratories participated. 

Graphical tools, such as the “target” plot to the left, have been used in individualized reports sent 

to each participant as well in the study’s format summary documents. Each comparability 

(difference from consensus) is estimated from results reported for seven different samples, 

centered on the sample’s consensus value and scaled to the interlaboratory standard deviation. 

Comparability has two aspects: concordance (average difference from consensus, plotted along 

the horizontal axis) and apparent precision (standard deviation about the average difference, 

plotted along the vertical axis). The 

best comparabilites are at the target’s 

center; the large red hole denotes a 

participant reporting values with 

excellent apparent precision and very 

good – if slightly low – concordance. 

One of the major findings from MSS3 

is that participants reporting DNA 

concentration measurements with 

unusually poor apparent precision or 

unusually high concordance values 

typically expended more analytical 

effort to produce DNA fingerprints of 

adequate quality. 

The third NIST-sponsored Mixed Stain 

Study (MSS3) was designed to help the 

forensic human identity community make 

more effective use of their limited 

resources by illuminating the relationships 

between the quality of their DNA 

concentration measurements and of their 

initial DNA “fingerprint” efforts. 
87

BIPM

9. Health and Medical Technologies 

CSTL provides quality assurance for the healthcare industry by providing 

the appropriate chemical and physical 

reference standards, developing 

reference methods and databases, and 

also by working closely with the clinical 

and medical community to transfer the 

accuracy of the highest level of 

metrology to high-throughput clinical 

laboratory measurements. Measurements are responsible for up to 15% of 

the $1.3 trillion annual costs of healthcare in the U.S. A significant portion (25% to 30%) of these 

measurements are performed for non-diagnostic reasons, such as re-tests, error prevention, and 

detection.

NIST Assists US IVD Industry in Meeting the Requirements of European Directive 98/79/EC 

W.F. Koch (830); and W. E. May and M.J. Welch (839) 

European Directive 98/79/EC on in vitro diagnostic medical devices (IVDs) requires that products 

be traceable to ìstandards of higher orderî, e.g., nationally/internationally recognized certified 

reference materials. It further states that such devices ìmust be designed and manufactured in 

such a way as to provide adequate 

stability and accuracy of measurement 

within appropriate accuracy limitsî and 

that ìthe accuracy limits have to be 

specified by the manufacturer.î 

Implementation date of this Directive is 

December 2003. At present, reference 

methods and/or reference materials are 

available for only a limited number of the 

several hundred analytes that are 

measured in medical 

laboratories on a 

daily basis using IVDs. Furthermore there is currently no internationally 

recognized procedure for stating accuracy claims in this arena. Since 

approximately 60% of the IVD products on the European market ($5.6 billion 

in 1998) are imported from the USA, this is a very important trade issue. 

In response to this EU directive and at the urging of the US medical device industry, NIST 

organized and hosted a special workshop on Measurement Traceability for Clinical Laboratory 

Testing in November 2000. Out of this workshop grew the concept and the need for an in 

international body to coordinate and prioritize the development of reference methods and 

reference materials for this industry (one of the few that still maintains a positive 

trade balance). The Joint Committee for Traceability for Laboratory Medicine, 

JCTLM (hosted by the BIPM) is the result. (NIST arranged and chaired a special 

pre-JCTLM meeting of US industry representatives to develop a coordinated US 

position on industryís response to metrologically traceable reference materials and 

methods for clinical analyses.) The main purpose of the JCTLM is to actively 

involve the IVD industry, professional organizations, government, and national and 

international standards developing organizations in the solution to establishing the means and 

processes for assuring measurement traceability and reference systems for the healthcare 

community. NIST played the pivotal role on the organizing and steering Committee for this new 

Healthcare costs amount to ~ 

14% of the GDP, an estimated 

$1.5 trillion. By 2007, healthcare 

spending as % of GDP is 

projected to reach over 16%. 

The recently enacted European Directive 

98/79/EC on in vitro diagnostic medical 

devices (IVDs) requires that products be 

traceable to “standards of the highest order”, 

e.g., nationally/internationally recognized 

certified reference materials (CRMs). Because 

approximately 60% of the IVD products on the 

European market ($5.6 billion in 1998) are 

imported from the USA, this is a very 

important trade issue. 
88

Structure of Cardiac Troponin C-Troponin I Complex

Image from PDB 

organization. After several preliminary and planning meetings, a full organizational meeting was 

held in Paris in June 2002. NIST was a proactive participant and was appointed chair of one of 

two working groups within the JCLTM. NIST hosted the first Working Group 1 meeting in August 

2003, actively engaging US industry involvement, and is proceeding in the planning of its second 

meeting in late March. 

Concurrent with these efforts within JCTLM, NIST is 

working closely with the US IVD industry in developing 

practical guidance on the calculation of the uncertainty of 

calibrator values for in vitro diagnostic manufacturers, in 

order to respond to the accuracy limits provision of the 

Directive. 

Standards for in vitro Diagnostics and International Trade 

W.E. May, D.M. Bunk, J.D. Fassett, W.R. Kelly, S.E. Long, K.E. Murphy, B.C. Nelson, M.B. 

Satterfield, L.T. Sniegoski, S. Tai, R.D. Vocke, and M.J. Welch (839) 

The accuracy and traceability of the data from medical tests are becoming increasingly important. 

Typically, medical guidelines are derived from clinical studies where medical outcomes are 

correlated with medical test results. Such data are 

often collected using many different laboratories 

and instruments, in different parts of the world, and 

at different times. However, effective use of such 

data will require that any differences observed be 

attributable to the clinical parameter(s) being 

measured and not to the measurement processes. 

In addition to reliability and cost concerns, there is an increased 

urgency for developing internationally accepted reference methods 

and certified reference materials for new diagnostic markers. 

ADVAMED, representing the U.S. IVD Industry, has asked NIST to 

work with our counterparts in Europe and the Asia-Pacific Region to 

provide the reference materials and methods of ìhigher orderî that are urgently needed to comply 

with the requirements of the EU IVD Directive, described in the previous article. Without timely 

completion of these standards, their access into the European market will be seriously 

jeopardized. Through the Joint Committee for Traceability for Laboratory Medicine (JCTLM), 

NIST, the EU and AP laboratories have agreed to share the workload involved in developing and 

maintaining the more than 100 standards needed for industry 

compliance with the EU IVDD. NIST has been asked to provide 

about 40% of the standards based on our current capabilities and 

U.S. market share. Currently NIST has developed and is 

maintaining high-accuracy reference methods for 26 clinical 

diagnostic markers including a number of electrolytes (calcium, 

chloride, lithium, magnesium, potassium, and sodium), hormones 

(cortisol, thyroxine) and small organic substrates (cholesterol, 

creatinine, glucose, urea, uric acid, 

triglycerides) to support the national reference system for clinical 

chemistry. Reference methods have also been developed and 

SRMs disseminated for a number of 

micronutrients (e.g., vitamins A, C, D, 

E, and beta-carotene) therapeutic drugs 

To respond to the accuracy limit 

provision of the directive, NIST 

organized and hosted a meeting 

of key industry representatives 

on January 7, 2003 to develop a 

short course on this topic to be 

webcast to the whole industry in 

early April. 

Valid decision-making requires that 

a medical test result for an 

individual patient – from a different 

laboratory at a later time – be 

correlated to the clinical study data 

for the broader population. 
89

A point-of-care test for 

troponin I levels following 

AMI. Photo from Spectral 

Diagnostics.

(dilantin, lithium, phenobarbital, theophylline, valproic acid) and drugs of abuse (cocaine, delta-9 

THC) as well. During the past year we have: (1) critically evaluated isotope-dilution ICP-MS as a 

replacement to isotope dilution thermal ionization 

mass spectrometry for serving as the U.S. reference 

method for electrolytes in blood serum, (2) developed 

and critically evaluated new reference methods for 

cortisol, folates, homocysteine, and triiodothyronine 

in blood serum, (3) certified the concentrations of Cd 

and Hg in whole blood (SRM 966) and completed 

analyses for value assignment of 10 toxic elements 

at trace and ultratrace levels in a new freeze-dried 

urine reference material, SRM 2670a and (4) 

evaluated six candidates to serve as a primary 

reference material for Cardiac Troponin-I (cTnI). 

Toward Comparable Cardiac Troponin I Measurements: Selection of a Primary Reference 

Material 

D.M. Bunk and M.J. Welch (839) 

The clinical measurement of serum cardiac troponin I (cTnI) has become an important tool in the 

diagnosis of acute myocardial infarction (AMI) and myocardial damage. Unfortunately, a ten-fold 

difference in measured concentrations among assays is 

common, and as much as a hundred-fold difference in 

response has also been observed. To address the need 

for standardization of cTnI assays, NIST, the American 

Association for Clinical Chemistry (AACC), and the 

International Federation for Clinical Chemistry (IFCC) have 

all collaborated to evaluate reference 

materials for clinical cTnI measurement. 

After evaluation of six different candidate reference materials using two round 

robin studies, a single reference material has been selected. More than 20 

different commercial cTnI assays, made by 12 different manufacturers, from the 

United States, Europe, and Japan, were used to evaluate the candidate 

materials. This choice was made after evaluation of the troponin preparation for 

purity, stability, its ability to provide harmonization of assay results, and the 

commutability of the material among the 20 commercial cTnI assays used in the study. A cTnI 

reference material will be used by the assay manufacturers to develop new assays and to 

validate calibration materials for their current assays. 

SRM 2921, purified human cardiac troponin complex, is currently 

being prepared. Once this material is available, a third round robin 

study will be 

carried out to 

assess the ability 

of SRM 2921 to 

harmonize the cTnI 

assay results from 

patient samples. 

Future work will 

also include the 

development of a 

reference method 

for cTnI in serum. 

NIST has developed and is maintaining 

high accuracy reference methods for 26 

clinical diagnostic markers. Additional 

reference methods and standards for 

new health status markers are in 

development. Improved accuracy 

facilitated by this program is leading to 

better diagnosis, treatment and reduced 

health care costs. 

In a recent issue of Clinical Laboratory News

(Vol 28, No 10, Oct 2002), the selection of a 

material to be used as a primary cTnI 

standard was touted as an ìimportant 

milestoneî in the standardization of clinical 

cTnI assays. Data from the second round 

robin demonstrates that the use of one 

common reference material could reduce 

interassay variability to 10 to15%, a 

substantial improvement compared to the 

130% variability observed in the first round 

robin. 

To reduce the large variation 

among results from clinical 

cTnI assays, NIST, in 

cooperation with the AACC 

and IFCC, is developing a 

cTnI reference material. 
90

New Reference Methods Developed for Cortisol, Folates, Homocysteine, and 

Triiodothyronine in Human Serum 

B.C. Nelson, M.B. Satterfield, L.T. Sniegoski, S. Tai, and M.J. Welch (839) 

The issuance of the in vitro diagnostic (IVD) device directive by the European Union has 

increased the need for NIST to develop new reference methods and SRMs to provide traceability 

for the US IVD industry so that this industry may 

maintain its strong position in European markets. 

In the past year, NIST has completed development of 

reference methods for the determination in blood of 

homocysteine, a new marker for risk of cardiovascular 

and other diseases; folates, critical vitamins for 

suppressing neural tube defects and the effects of 

elevated homocysteine; triiodothreonine (T3), a marker 

for thyroid function; and cortisol, a hormone important to 

endocrine function. These new methods were based upon isotope dilution combined with liquid 

chromatography/mass spectrometry (LC/MS), LC with tandem mass spectrometry (LC/MS/MS), 

and gas chromatography/mass spectrometry (GC/MS). The LC/MS method for folate and the 

LC/MS, LC/MS/MS, and GC/MS methods for homocysteine developed at NIST were extensively 

evaluated and tested against methods used by the Centers for Disease Control and Prevention 

(CDC) and elsewhere and found to provide results of high accuracy and precision. The 

LC/MS/MS method for T3 along with an LC/MS/MS 

method for tetraiodothreonine (T4) was used in an 

international comparison with excellent agreement with 

laboratories in Europe. The LC/MS/MS method for 

cortisol has been thoroughly tested and it provides 

unrivaled precision and specificity for this low-level 

hormone. 

Newly Certified Trace Elements in Urine and Blood SRMs 

R.D. Vocke, C. Beck II, O. Ertas, W.R. Kelly, S.E. Long, and M.S. Rearick (839) 

Noninvasive methods for obtaining information on human biological function are useful both as 

screening tools for population studies and for individual diagnoses of medical disorders. Urine 

and blood are the two most common body fluids used in clinical evaluations of selected trace and 

ultra-trace element levels in humans. Besides 

the obvious importance of toxic element 

markers as indices of occupational exposure 

(e.g., Pb, Cd, Hg), there is increasing interest 

within the medical profession of the precious 

metal levels in such fluids and how that might 

be tied to surgical implants as well as exposure 

to automotive exhaust that has passed through 

catalytic converters. The Centers for Disease 

Control and Prevention (CDC) is also 

increasing the frequency of their health surveys in order to establish population baselines for a 

wide range of elements and releasing these values directly to the public. Such measurements 

are of great interest to public health officials but are very difficult to make because of the 

extremely low level of the analytes and are in need of a quality control (QC) material. Our effort 

in characterizing urine and whole blood materials has been directed particularly towards certifying 

the elements within the low or normal levels, which have typically been listed as ìinformation 

valuesî due to the difficulties in making measurements near the limits of quantitation. We have 

NIST has a strong commitment 

to promoting accuracy in 

health-related measurements 

and providing measurement 

traceability to the U.S. in vitro 

diagnostic (IVD) industry 

through development of 

reference methods and SRMs.

These new reference methods and 

the SRMs that they will be used to 

certify will provide critical 

traceability to the IVD industry and 

will help improve the reliability of 

routine clinical measurements. 

Clinical analyses are used both as tools 

for screening the state of health of a 

given population as well as for individual 

diagnoses of medical disorders. Urine 

and blood are the two most common 

body fluids used in clinical evaluations of

selected trace and ultra-trace element 

levels in humans. 
91

focused on two reference materials, SRM 966 (toxic elements in frozen whole bovine blood) and 

SRM 2670a (trace elements in urine). 

We have completed certification analyses for 

Cd and Hg in the base level of the whole 

blood reference material, SRM 966, utilizing a 

combination of a new technique and 

instrumentation. 

Using an extremely sensitive Hg isotope 

dilution-hydride generation-ICPMS technique, 

a certified Hg value of (44.5 ± 3.3) ng/L was 

successfully measured in the low level of this 

material. Cadmium was also re-measured 

using the high sensitivity of the sector-field 

ICPMS together with isotope dilution mass 

spectrometry culminating in a certified Cd 

value of (31.8 ± 18) ng/L. 

Both elements in this material had previously been 

ìinformation valuesî for Hg and Cd at < 60 ng ng/L and < 

400 ng/L, respectively. We have also completed analyses of 

10 elements at trace and ultratrace levels in a new freeze 

dried urine reference material, SRM 2670a. Selected data 

are shown in the plot, comparing the values in the new 

material with the information values (where they existed) in 

SRM 2670. 

Critical Evaluation of ID ICP-MS vs. TIMS as New National Reference Method for 

Electrolytes in Blood Serum 

K.E. Murphy, S.E. Long, J.D. Fassett, and M.S. Rearick (839) 

Reference methods play a critical role in the measurement infrastructure required to diagnose, 

treat and prevent disease. Decisions regarding health status are based on measurements of 

electrolytes, such as potassium (K) and calcium (Ca). The accuracy of these measurements 

depends on accessible reference methods and 

standards. We have developed a new reference 

method for the determination of K and Ca based on 

ìcold plasmaî isotope dilution inductively coupled 

plasma mass spectrometry (ID ICP-MS). Traditionally, 

isotope dilution thermal ionization mass spectrometry 

(ID TIMS) has provided the accuracy base and SI 

traceability for the determination of these analytes in 

clinical materials. ICP-MS is simpler to implement and more widely available than TIMS. We 

benchmarked the cold plasma ID ICP-MS methodology against the previously applied ID TIMS 

definitive method in several ways. First, a direct comparison was performed by using both 

techniques for ratio measurements of K and Ca on the same isotope dilution samples. Second, 

an indirect comparison was made through the analysis of Ca in Standard Reference Material 

956a Electrolytes in Frozen Human Serum, which had been certified by ID TIMS in 1996. Third, 

we demonstrated comparability for serum Ca results with other National Metrology Institutes 

(NMIs) that applied ID TIMS through participation in the Consultative Committee for Amount of 

Substance (CCQM) Pilot Study, P-14. All sources of bias in the method were considered and a 

complete uncertainty statement was given. The ID ICP-MS expanded uncertainty based on a 95 

% confidence interval for the determination of Ca in P-14 serum samples was ± 0.42 % relative. 

The application of new 

instrumentation and analytical 

techniques has led to an 

increase in the number of 

elements that are being 

certified compared to earlier 

clinical SRMs. 

High-accuracy reference 

methods for electrolytes in body 

fluids play a critical role in the 

measurement infrastructure 

required to diagnose, treat, and 

prevent disease. 

Al

2670a - Low 2670 - Low 2670a - High 2670 - High

[ng/mL] 0.001

0.01

0.1

1

10

100

1000

Th

U

Pb

Tl

Cd

Hg

I

Mn

V

(Pb)

(Cd)

(Hg)

(Al)

(Mn)

Th

U

Pb

Tl

Al

Hg I

Mn V

Pb

Cd Hg

(Al)

(Mn)

(V)

Symbol Legend

(Mn) Information Value

 Mn Certified Value

Summary of Selected Trace Element 

Concentrations in Urine SRMs 
92

The critical evaluation of this ICP-MS-based procedure 

and our demonstrated ability to achieve comparability 

with a definitive method meets the demand for 

accurate and accessible clinical measurement 

methodology and satisfies traceability and global 

comparability needs. 

Measurement Quality Assurance Program for Fat-Soluble and Water-Soluble Vitamins in 

Blood Serum 

J.M. Brown Thomas, D.L. Duewer, S.A. Margolis, and K.E. Sharpless (839); and M.C. Kline 

(831) 

The Micronutrients Measurement Quality Assurance (QA) Program supports measurement 

technology for selected fat-soluble and water-soluble vitamins and carotenoids in human serum 

and plasma. This program was initiated in 1984 in conjunction with the National Cancer Institute 

(NCI) Division of Cancer Prevention and Control to 

ensure the long-term reliability and comparability of 

the measurements made while studying the possible 

cancer chemoprevention role of these analytes. From 

an initially NCI-supported core of 10 NCI-grantee 

laboratories, the program currently involves 45 feepaying laboratories worldwide. This program provides 

participants with measurement comparability 

assessment through use of interlaboratory comparison studies, Standard Reference Materials 

(SRMs) and control materials, and methods development and validation. Significant 

improvements in measurement comparability among QA participants have been documented. 

Many of the participants depend on this QA program to demonstrate their compliance with 

Clinical Laboratory Improvement Act (CLIA) requirements. 

Serum-based samples with assigned values for 

target analytes (e.g., retinol, tocopherols, ∃-

carotene, and ascorbic acid) and performanceevaluation standards are distributed by NIST 

biannually to participants for analysis. NIST 

provides feedback to the participants 

concerning their measurement performance 

and suggestions for methods development and 

refinement. The comparability of laboratory 

measurements for target analytes has 

improved substantially over time. This 

improvement has been achieved through the 

development and promulgation of robust 

measurement technologies, identification and 

production of suitable reference materials, 

isolation and identification of measurement 

system biases, and support and 

encouragement of long-term within-laboratory 

measurement quality control efforts. The long-term 

stability of the target analytes in lyophilized and 

liquid-frozen reference materials stored at -80 °C has 

been documented, as has the utility of a variety of 

graphical tools for the analysis of interlaboratory 

measurements. More information about the QA 

program is available on the web. 

Development of a reference method 

based on ICP-MS takes advantage of 

modern, widely available instrumentation 

and realizes a factor of four improvement 

in the uncertainty interval, approaching 

the capabilities of the definitive method. 

The Micronutrients Measurement 

QA Program is designed to assess 

and improve the comparability and

reliability of measurements of 

vitamins and carotenoids in 

human serum and plasma. 

Results from the interlaboratory 

comparison studies help 

participants in the QA program make 

accurate clinical and health-care 

decisions and maintain and improve 

their measurement comparability.

http://www.cstl.nist.gov/nist839/839.02/qa.html
93

The Complete Mitochondrial DNA (mtDNA) Genome Sequence of Human Cell Line HL-60 

and Its Inclusion in NIST’s mtDNA SRM 

B.C. Levin, D.K. Hancock, K.L. Richie (831); K.A. Holland (Gettysburg College); M. Coble 

and T. J. Parsons (Armed Forces DNA Identification Laboratory); L.J. Kienker (FBI 

Laboratory); and D.W. Williams (Georgia Bureau of Investigation) 

 

MtDNA is used by the forensic community for human identification and by the medical community 

for diagnoses of human mtDNA diseases. An mtDNA Standard Reference Material (SRM 2392) 

prepared by NIST to provide quality control to the scientific and clinical communities when they 

amplify and sequence human mtDNA became available in 

December 1999. This SRM includes two human DNA 

templates (CHR and 9947A) and all the information 

necessary to successfully conduct PCR amplification, cycle 

sequencing, gel separation, and data analysis to obtain the 

final DNA sequence. The sequences of 58 unique primer 

sets, which allow the sequencing of the entire mtDNA 

(16,569 bp) with no gaps, are also provided. The FBI 

recently requested that NIST add DNA from cell line HL-60 

which has several evenly spaced polymorphisms in the 

mtDNA control region and no C-stretch (difficult to sequence) areas. The FBIís 

Combined DNA Index System (CODIS) program now includes mtDNA 

sequences from unidentified remains, as well as from relatives of missing 

persons. In order for authorized laboratories to contribute or examine these 

indices, the FBI has deemed that certain quality standards must be met. In 

particular, a positive control from the human cell line HL-60 must be run in 

conjunction with their unknown samples. This addition of 

HL-60 to the NIST SRM 2392 and an interlaboratory 

evaluation of the HL-60 sequence by three other 

laboratories have been completed. The new SRM 2392a 

will provide additional quality control when amplifying and 

sequencing human mtDNA. Corroboration of the SRM 

results will provide assurance that unknown DNA 

samples are also being amplified and sequenced 

correctly. The primary customers are the forensic laboratories throughout the U.S. and the clinical 

laboratories that screen the DNA of patients for mitochondrial diseases. 

Mitochondrial DNA Mutations in Patients with Myelodysplastic Syndromes 

B.C. Levin (831); and M.G. Shin, S. Kajigaya, and N.S. Young (NIH) 

Recently, a number of papers have been published that equate mtDNA mutations with specific 

diseases, including cancer, which had previously not been associated with mtDNA mutations. 

The functional relevance of these mutations (i.e., whether they have a causal relationship or 

could serve as biomarkers for early detection of the disease) has not been proven. In this 

research, we examined the mtDNA mutations found in 

patients with Myelodysplastic Syndrome by 

amplification and sequencing of the entire human 

mtDNA (16569 bp) and compared the results with a 

comparable normal control group. The entire mtDNA 

genome (16,569 bp) in 10 patients with Myelodysplastic 

Syndrome and 8 normal controls was amplified by PCR 

and sequenced. The sequences were analyzed and 

compared to each other as well as recognized 

mitochondrial DNA databases. Overall, there was no increase in the number of mtDNA genes 

DNA SRMs provide quality 

control and assurance that 

laboratories are sequencing

unknown DNA samples 

correctly. 

The inclusion of HL-60 DNA in SRM 

2392a will enhance this SRMís 

utility to forensic laboratories, to 

clinical laboratories that screen 

patients for mtDNA mutations and 

diseases, and to toxicologists who 

screen mtDNA for mutations. 

Over 100 point mutations and 

more than 200 deletions and 

rearrangements in mtDNA have 

been associated with disease, 

and new mutations are being 

described every year. However, 

the database of "normal" mtDNA 

sequences is relatively limited. 
94

harboring polymorphisms or ìnewî mutations between our patients and normal controls, although 

there were a few more mtDNA changes resulting in amino acid changes in myelodysplasia (16 in 

10 patients versus 9 in 8 controls). Thirty new mutations, all nucleotide substitutions, were found 

among the ten patients, distributed throughout the mitochondrial genome; five mutations resulted 

in amino acid changes, but none in the controls. We were not able to confirm previously 

described mutations in sideroblastic anemia, nor ìhot spotsî in the Cytochrome C Oxidase I and II 

genes. Our data do not support a major role for mitochondrial genomic instability in 

Myelodysplasia, and they fail to confirm previous 

reports of significant or widespread mitochondrial 

mutations in this disease. Modest changes in 

mutation numbers and mitochondrial microsatellites 

may be evidence of increased mutagenesis in 

mtDNA, or, more likely, a reflection of limited clonality 

among hematopoietic stem cells in this bone marrow 

failure syndrome. 

The Role of the Cockayne Syndrome Gene in Cellular Repair of Oxidative DNA Damage 

M.M. Dizdar, H. Rodriguez (831); J. Tuo and V. Bohr (NIH); and P. Jaruga (UMBC, MD) 

Cockayne Syndrome (CS) is a human genetic disorder with diverse clinical symptoms that 

include hypersensitivity to sunlight, severe mental and physical growth retardation, progressive 

neurological and retinal degeneration, and skeletal abnormalities. CS has two complementation 

groups, CS-A and CS-B. The gene of CS-B (CSB gene) 

encodes a protein (CSB protein), which is known to play a 

role in the cellular repair of DNA damage, but it may 

participate in other pathways of DNA metabolism. In this 

study, the role of the CSB protein in the cellular repair of 

oxidative DNA damage was investigated.

Transformed human cell lines with site-directed mutations in the CSB gene were established. 

These cell lines were then used to study phenotypical changes affected by the mutations, 

including DNA repair by whole cell extracts and 

the accumulation of oxidative DNA damage in 

genomic DNA of cells after exposure to oxidative 

stress. Cells were exposed to low doses of 

ionizing radiation such as 2 Gy to cause oxidative 

stress. It was found that the mutant cells and 

those with the deleted CSB gene had greater 

sensitivity than wild-type cells to ionizing 

radiation. The mutant cells had lower activity of 

DNA repair as shown by determination of 

activities of DNA repair enzymes. The results 

showed that the biological functions of the CSB 

protein in DNA repair might be mediated by 

distinct functional motifs of the protein. In 

addition, the accumulation of two well-known 

products of oxidative DNA damage, i.e., 8-

hydroxy-2¥-deoxyguanosine (8-OH-dGuo) and 8-

hydroxy-2¥-deoxyadenosine (8-OH-dAdo) in 

genomic DNA of cells was measured using the 

technique of liquid chromatography-mass 

spectrometry. These products accumulated to a greater extent in mutant cells and in cells with 

the deleted CSB gene than in wild-type cells after exposure of cells to ionizing radiation. This 

This work points out the importance of 

preparing a database of the sequences 

of the entire mtDNA of normal 

individuals such as those that are 

available in the NIST SRM 2392. 

Without such controls, the importance 

of mtDNA polymorphisms and 

mutations may be overestimated. 

Accumulation of 8-OH-dGuo and 8-

OH-dAdo in genomic DNA of 

Cockayne Syndrome patients after 

exposure to γ-radiation

Studies of DNA damage 

can provide clues to the 

pathogenesis of human 

diseases. 
95

work was then extended to primary fibroblasts from eleven CS 

patients and six control individuals. These cells were exposed 

to 2 Gy of ionizing radiation to induce oxidative DNA damage 

and were then allowed to repair the damage. No significant 

change in background levels of the aforementioned 

compounds was observed in DNA of fibroblasts of normal 

individuals, indicating complete cellular repair. In contrast, 

cells from CS patients accumulated significant amounts of 

these lesions, providing evidence for a lack of DNA repair. 

Design and Use of a Peptide Nucleic Acid (PNA) for the Detection of the Heteroplasmic 

Low-Frequency MELAS Mutation in Human Mitochondrial DNA 

D.K. Hancock, F.P. Schwarz, B.C. Levin (831); F. Song (CARB/UMBI); and L.-J.C. Song 

(Georgetown University Medical Center) 

Most pathogenic human mtDNA mutations are heteroplasmic (i.e., the mutant mtDNA coexists 

with the normal mtDNA in the tissue or cell). The mutation level varies with the tissue and is often 

difficult to detect (especially in blood samples) when very low levels of the mutant exist in a 

population of wild-type mtDNA molecules. 

We are using a simple methodology to 

detect low levels of the single base pair 

heteroplasmic MELAS (Mitochondrial 

Encephalomyopathy, Lactic Acidosis, and 

Stroke-like Episodes) (A3243G) mutation. 

We designed, synthesized, characterized, 

and purified a series of PNAs and tested 

them to determine the best one and the 

optimal conditions for blocking the 

amplification of the wild-type mtDNA while 

allowing the mutant DNA to amplify. We 

determined the optimal concentration of the PNA to be about 2 μmol/L. We examined eight 

MELAS patients as well as control healthy individuals. In all the MELAS patients, we were able to 

show that mutations that were not visible or barely visible in the sequence electropherogram in 

the absence of PNA became the dominant peak and 

readily detectable in 

the presence of our 

designer PNA. The 

lowest detectable 

level of the mutation 

was 0.1%.

The results of this study will have a 

considerable impact on the clinical 

community who screen patients for 

mitochondrial diseases. It will greatly 

facilitate the detection of a mutation 

that was previously extremely difficult 

to detect and will help to prevent false 

negative results. 

Future Plans: NIST plans to develop an SRM for the detection of the MELAS 

mutation in human mtDNA. This SRM will consist of the specially designed PNA, 

the primers needed to amplify the region where the mutation occurs, and the 

optimized protocol to allow any laboratory with PCR and sequencing capabilities 

to use this SRM and detect the MELAS mutation in blood even if it is not 

detectable in the absence of the PNA. 

A multitude of mitochondrial DNA (mtDNA) 

diseases have been correlated with 

single nucleotide polymorphisms (SNPs), 

mutations, insertions, and deletions. Most of 

these diseases are neuromuscular, but 

deafness, diabetes, epilepsy, progressive 

dementia, hypoventilation, cardiac 

insufficiency, renal dysfunction, and sudden 

onset blindness are some of the other 

symptoms of mtDNA mutations. 

The results of this study 

suggest that the accumulation 

of oxidative stress-induced 

DNA lesions in genomic DNA of 

the CSB patients may 

contribute significantly to the 

pathogenesis of the human 

disorder CS. 
96

Expression and purification of NEH1. Lysates (15 μg) of E. coli

expressing NEH1 were analyzed by SDS/PAGE and Coomassie 

blue staining. The Bio-Rad protein markers are shown on the left. 

Purified WT NEH1 (lane 1); lanes 2-4, lysates of E. coli with empty 

vector, WT, and Pro-1 mutant NEH1 expression plasmid, 

respectively. The position of NEH1 is indicated by arrow. 

Discovery of a Critical DNA Repair Enzyme 

M.M. Dizdar (831); P. Jaruga (UMBC, MD); and T. Hazra and S. Mitra (Univ. of Texas) 

The identification and characterization of DNA 

repair enzymes might be the first step toward 

understanding how oxidative stress produces 

health problems and/or exacerbates the existing 

diseases. This study was conducted to determine 

the specificity of one important human DNA 

repair enzyme, NEH1, using mass spectrometric 

techniques. In the human genome database, 

sequences were identified that are similar to 

those known to code for DNA repair enzymes in 

the bacterium Escherichia coli. Following identification of the sequences, a previously unknown 

protein was produced. This protein was characterized by various techniques. Mass spectrometric 

measurements proved this enzyme to function as a DNA glycosylase when tested on oxidatively 

damaged DNA in vitro, and to remove two biologically 

important lesions from DNA. DNA glycosylases are repair 

enzymes that are involved in the first step of a complex 

DNA repair process in 

cells and remove 

modified bases from 

DNA. This glycosylase 

named NEH1 possesses 

two characteristics. First, 

it works on critical 

portions of human DNA 

that are actually active. Second, when DNA is replicating, cells 

produce more NEH1 as if in an effort to repair errors caused by 

oxidative DNA damage before they are fixed in the next 

generation causing mutations. This coupling of repair to DNA 

replication indicates that the repair of active genes is different 

from the repair of the bulk of the genome. NEH1 was found to be 

highest in the liver, pancreas, and thymus. The paper that 

describes these results was published in Proceedings of 

the National Academy of 

Sciences and featured on the 

cover page of this journal. 

Oxidative DNA damage results from 

normal cellular metabolism and 

oxidative stress, and is implicated in 

numerous human diseases from 

cancer to neurodegenerative 

diseases such as Alzheimer’s 

disease to the normal aging process.

Detailed knowledge of mechanisms 

of DNA damage and repair might 

lead to drug developments and 

clinical applications including the 

improvement of cancer therapy by 

inhibiting DNA repair in drug- or 

radiation-resistant tumors and/or 

the increase in the resistance of 

normal cells to DNA damage by 

overexpressing DNA repair genes. 
97

DNA and Protein Gel Plugs for Biological Assays 

R.A. Zangmeister, G.A. Thomas, D.J. Ross, and M.J. Tarlov (836); and K. Olsen (Loyola 

College) 

CSTL has devised a new, versatile method for 

securing biological ligands in microfluidic devices for 

a variety of biomedical assays. One such assay 

involves hybridization of nucleic acids for DNA 

sequence determinations. Single stranded DNA 

(ssDNA) probes modified on one end with an 

acrylamide functional group are photo-polymerized with polyacrylamide, creating a stable gel 

matrix with the ssDNA covalently bound. Complementary single stranded sequences, or targets, 

that are electrophoretically driven through such probe-containing gel matrices are captured, while 

non-complementary targets migrate through. 

The high concentration of the probes in the gel plugs 

coupled with the enhanced mass transfer characteristics 

of microfluidic channels ensure that targets will 

encounter a complementary probe, be captured, and 

thus be detected. Spatially distinct gel plugs each 

containing a different probe sequence can be formed 

photo-lithographically in a single channel for 

simultaneous detection of multiple targets. 

In addition, a parallel effort this year demonstrated a 

conceptually similar approach for antigen capture assays 

where antibodies were entrapped in photo-polymerized 

gel-plugs. Specific antibody/antigen interactions using 

target and non-target antigens were demonstrated with 

the hydrogel-entrapped antibodies. 

Future work is expected to demonstrate enhanced utility of gel-plugs to applications involving 

catalytic DNA. A novel two-plug probe/capture 

strategy will be used in conjunction with 

catalytic DNA for the detection of Pb in water. A 

paper describing the formation and use of DNA 

gel plugs was published in Analytical Chemistry 

in 2002. 

A new method for immobilizing biological 

capture ligands in microfluidic devices has 

been devised with potential for use in 

disease diagnosis, and high throughput 

genomics and proteomics. 

Researchers are developing 

techniques to improve the 

analytical sensitivity of microfluidic 

devices for biomedical assays. 

Scheme for competitive DNA assay using probe (1st

plug) and capture (2nd plug) gel plugs. Plots of 

fluorescence intensity measured of tagged 10mer 

initially hybridized in gel plug 1 during electrophoresis 

of both complementary and non-complementary 20mer 

targets. 

0.0 1.0 2.0 3.0 4.0

0.00

0.25

0.50

0.75

1.00

Time (min)

 1st Plug

 2nd Plug

0.00

0.25

0.50

0.75

1.00

 Control rinse

Normalized Fluorescence Intensity of 10mer Target

 1st Plug

 2nd Plug

20mer

tagged 10mers

1st plug 2nd plug

Non-complementary trial

Complementary trial
98

Temperature Gradient Focusing: A New Preconcentration and Focusing Technique for 

Microfluidic Devices 

D. Ross (836); and L.E. Locascio (839) 

One of the remaining problems in the field of microfluidics is the need for an effective method of 

pre-concentration that is suitable for miniaturization and integration into ìlab-on-a-chipî platforms. 

A new focusing method, Temperature Gradient Focusing (TGF), has been developed to meet the 

outstanding need for effective pre-concentration 

techniques for microfluidics. Initial experiments 

indicate that the new method has great promise 

as a technique for preconcentration as well as 

for the focusing and separation of a variety of 

charged analytes including fluorescent dyes, 

amino acids, proteins, DNA, and colloidal 

particles. TGF operates by balancing the 

electrophoretic motion of analytes in a micro-channel against the bulk flow of buffer solution 

through the channel while applying both an electric field and a temperature gradient along the 

channel. If the ionic strength of the buffer solution is temperature-dependent, the temperature 

gradient will give rise to a corresponding gradient in the electrophoretic velocity of a charged 

analyte in the channel. The bulk flow velocity can then be adjusted so that the total analyte 

velocity (the sum of the bulk and electrophoretic velocities) is zero at some point along the 

channel, and all of the analyte will be focused at that point. As a method for analyte preconcentration, TGF out-performs all other 

methods, with demonstrated concentration 

factors exceeding 10,000. In addition to analyte 

pre-concentration, TGF can be used for the 

separation of different charged analytes in a 

manner analogous to isoelectric focusing of 

proteins. TGF is much simpler to implement than 

isoelectric focusing and has the additional 

advantage that it can be used with any charged 

analyte, rather than just proteins. This versatility 

is shown graphically in the figure with 

separations of a variety of analytes. 

 

CSTL scientists have devised a new 

approach to pre-concentration for 

microfluidic devices. This method has 

demonstrated concentration factors 

exceeding 10,000 and can be used for a 

wide variety of charged analytes. 

The TGF method is relatively simple 

compared with other pre-concentration 

methods, making it particularly well suited 

to integration into ìlab-on-a-chipî devices.

Demonstration of TGF for the focusing 

and separation of (from top to bottom) 

small fluorescent dyes, amino acids, 

proteins, DNA, and colloidal particles. 
99

10. Industrial and Analytical Instruments and Services 

Instrument manufacturers depend on 

NIST/CSTL for physical and chemical 

standards, as well as NIST 

databases, for instrument calibration 

to provide traceability to national 

standards to the end users of their products and instruments. CSTL 

work supports both mature analytical applications, as well as the 

newest technologies such as high-throughput screening, and 

cutting edge research for next-generation semiconductor 

manufacturing.

Atomic Standard of Pressure 

M.R. Moldover, and J.W. Schmidt (836); and K. Szalewicz (Univ. of Delaware) 

Below 300 kPa, the primary pressure standard at NIST is a mercury manometer. Above 300 kPa, 

piston gauges, commercially manufactured piston-cylinder sets, are the practical widely-used 

pressure standard achieving measurement uncertainties of a few parts per million (ppm or 

Pa/MPa). Continued performance improvement necessary to achieve measurement uncertainties 

significantly below 1 ppm appears to be problematic. 

Therefore, a new approach to primary pressure 

measurement standards above ≈ 300 kPa must be 

developed if improved performance levels are to be 

realized. This will be accomplished both by measuring 

and calculating the dielectric constant ε(p,T) of helium 

with extraordinary accuracy and will permit the 

determination of the pressure p(ε,T) from electrical and 

temperature measurements of helium. The calculation of 

ε(p,T) from quantum mechanics by several theorists has 

improved dramatically recently with the variation 

between the results of all calculations falling below the 1 ppm level. The probability of continued 

improvement within the next few years is quite good. 

Dielectric constant measurements are being improved by drawing on NISTís expertise in 

electrical metrology to develop a novel, doughnut-shaped, four-electrode, cross capacitor as a 

pressure cell. In general cross capacitors are extremely stable electrically and far less subject to 

surface contamination (oxides, adsorbed water, or films of oil) when compared with capacitors 

using other geometries. The toroidal design has the further advantage of eliminating spurious 

end effects that complicate the measurements. During FY01/02, the cross capacitor and 

pressure vessel were tested at 0°C, 30°C, and 50°C by measuring the dielectric constant of 

helium. All of the measured values of ε(p,T) from two separate cross capacitors of differing 

designs were consistent with the theoretical values and with each other. 

By 2004, U.S. shipments of 

industrial and analytical 

instruments are expected 

to total nearly $60 billion. 

CSTL researchers are 

developing a novel primary 

standard for pressure in the 

range 0.3 MPa to 5 MPa. This 

is accomplished by both 

measuring and calculating the 

dielectric constant ε(p,T) of 

helium with extraordinary 

accuracy. 

CT B

C IO 

Toroidal Cross Capacitor and Component
100

CSTL has also used cross capacitors to 

measure the dielectric constants of the 

primary constituents of natural gas including 

methane, ethane, propane, nitrogen, carbon 

dioxide, and argon. The measurements 

span the 0 oC to 50oC temperature range 

and extend to 7 MPa. In this range, these 

data are more accurate than any previous 

measurements and provide reference data for use in metering natural gas based on heating 

value. In fact, they are of sufficient accuracy, better than 0.1%, to support new approaches to online measurement of the heating value of natural gas with significantly greater precision and 

accuracy than current methods based on chromatographic constituency determination. 

Bevel Craters for Surface Analysis of AlGaAs/GaAs Multilayer Structures 

S.A. Wight, J.G. Gillen, and A.J. Fahey (837) 

The lateral wet-thermal oxidation of high aluminum content AlGaAs layers can be used to form 

buried oxide layers that are widely employed for the fabrication of vertical cavity surface emitting 

laser structures used in the optoelectronics 

industry for fiber optic data links, proximity 

sensors, encoders, laser range finders, laser 

printing, bar code scanning, and optical 

storage. The challenge is to analyze the 

variation in composition of the oxidized layers 

laterally and as a function of depth. The oxidized 

portions of the AlGaAs layers ion beam etch much 

more slowly than the un-oxidized AlGaAs and the 

interstitial GaAs layers. This complicates depth profile 

measurements by Auger or secondary ion mass 

spectrometry (SIMS). A commercial secondary ion 

mass spectrometer (SIMS) instrument was modified 

with a custom external raster waveform. This 

waveform was designed to deliver a variable ion dose. 

The desired result was a bevel crater, one that ramps 

down from the top surface at one edge to some depth 

at the other edge. This produces lateral magnification 

of the subsurface structure for analysis by static SIMS 

or scanning Auger spectroscopy. The diagonal slice 

through the AlGaAs/GaAs multi-layers exposes a 

larger portion of the oxidized AlGaAs layer for 

subsequent analysis. The raster profile, ion species, 

and ion energy were evaluated by scanning electron 

microscopy and Auger imaging in a commercial UHV 

Auger microscope. Oxygen was chosen over cesium 

and gallium as the primary ion species because of the 

decrease in differential sputtering. The lower energy 

(3 keV) oxygen was selected over higher energies 

because it reduced surface roughening. Low energy 

argon (1 keV) ion sputter cleaning is being used in the 

Auger microscope to remove the primary sputter ion 

species because oxygen is an important component in 

the elemental analysis. 

This work has the potential to provide the basis 

for improved determination of the Boltzmann 

constant. Near-term impacts derive from the 

significantly improved measurement of 

dielectric constant for a range of gases, notably 

the primary constituents of natural gas. 

Perpendicular Cross-Section

Top Bevel Section

Secondary electron image of bevel 

exposed oxidized AlGaAs layers. 

Red lines follow one layer from side 

section to top bevel section 

The development of the bevel crater 

has facilitated the analysis of the 

oxidized AlGaAs. The bevel crater 

technique has also been successfully 

applied to other multi-layer structures,

such as Ni/Cr depth profile standard 

and KRISS sample. 

CSTL scientists are investigating 

improved techniques for characterization 

of multi-layer structures used in the 

optoelectronics industry. 
101

Chemical Imaging of Polymer Blends with Confocal Raman Microscopy 

C.A. Michaels (837) 

Confocal Raman microscopy has been used to study spatial variations in chemical composition 

and crystallinity for polymer blend films of interest to the industrial members of the NIST/industry 

polymer interface consortium (Dow Chemical, PPG 

Industries, Visteon, and MTS Systems). This 

chemical imaging technique yields data sets 

consisting of Raman spectra at each (x,y) sample 

coordinate, with diffraction limited lateral resolution (≈

0.5 μm) and excellent depth discrimination (≈ 5 μm). 

The chemical selectivity of Raman spectroscopy has 

been exploited to characterize the spatial distribution 

of a high value fluoropolymer in an acrylic resin film 

used as a protective coating for metal substrates in 

building applications. Raman spectra at 785 nm excitation of the film/substrate and film/air 

interfaces of a blend film (50/50 mass ratio) suggest a preferential depletion of (polyvinylidene 

fluoride) PVDF at the substrate interface. However, images of film cross sections, as shown in 

Figure 1(a), where the intensity of the CF2 stretching band at 799 cm-1 has been plotted, reveal 

no obvious PVDF gradient between the 

film surfaces. The markedly different 

surface and air interface spectra, coupled 

with the lack of a strong CF2 gradient in the 

film cross sections, suggest that only a 

thin, surface depletion layer exists at the 

substrate interface. figure (b) is a 

fluorescence image of the film cross 

section recorded simultaneously with that 

shown in figure (a), the difference in the 

images clearly demonstrating the chemical 

selectivity of Raman imaging. The sample 

preparation method used here is expected 

to result in significant surface roughness, 

and measurements designed to assess its 

impact on the spectral maps are ongoing. 

This technique has also been used to examine the relationship between thermal history and film 

morphology through spectral characterization of the level and spatial variation in crystallinity for 

polyethylene (PE) blends. A CH2 bending vibration at 1418 cm-1 results from a factor group 

splitting due to the crystal field of the orthorhombic crystal structure and is a clean signature of 

crystallinity in polyethylenes. Mapping of this band intensity (normalized to an internal standard) 

yields a crystallinity image with diffractionlimited lateral resolution and depth resolution 

set by the confocal parameter of the collection 

optics, typically on the order of 5 μm. Current 

work is focused on samples for which the 

thermal processing has been optimized for 

production of crystallites with 1μm to 20 μm 

diameters. 

CSTL scientists are examining 

chemical composition and 

crystallinity of polymer blends of 

interest to industrial collaborators 

in the NIST/industry consortium on 

the Characterization and Modeling 

of the Interfaces and Interphases of 

Polymeric Materials and Systems. 

This work was recently presented at a 

consortium review meeting, held at PPG 

Industries Coatings R&D Center in Allison 

Park, PA. Efforts to facilitate the transfer of 

confocal Raman chemical imaging technology 

to other industrial partners through sample 

evaluation and interaction with visiting 

scientists are ongoing. 

(a) 64x64μm intensity map of CF2 Raman band 

of PVDF/PMMA-co-PEA film cross-section. The 

film/substrate (air) interface is on the right (left) 

side of the film cross section. (b) Fluorescence 

intensity map acquired simultaneously with (a). 

a b 
102

Development of High-Precision Isotope Reference Standards 

A.P. Miiller (836) 

Although conventional K/Ar dating methods are widely used for dating minerals and geologic 

materials, significant interlaboratory disagreements (1 to 2%) frequently exceed intralaboratory 

accuracies by an order of magnitude. To improve calibration of the mass spectrometers used, 

several high-precision argon isotope spike systems have 

been developed for the U.S. Geological Survey, each 

consisting of a reservoir and a Dorflinger pipette capable 

of delivering the required (2 to 4) x 10-10 moles per 

aliquot. Two systems will deliver pure 38Ar and three will 

deliver artificial mixtures of 40Ar/36Ar with uncertainties 

that are expected to be 0.1 to 0.2 % for both amount of 

gas delivered and for isotopic ratios of the mixtures. The 

performance of these systems exceeded USGSís 

expectations by at least a factor of two. The ultra-low fill 

pressures (≈10 Pa) proved to be the major challenge in 

calibrating the spike systems with requisite accuracy. 

The MMhb-2 will be certified by CSTL for potassium 

and argon concentrations and distributed as an 

SRM, which will be used by argon dating laboratories 

along with the spike systems to resolve longstanding 

differences. The spike systems will also enable 

USGS to re-measure the isotopic composition of 

atmospheric argon and calculate a new atomic 

weight for argon. 

The argon spike systems will be used by 

USGS to measure the argon concentration 

of a new preparation of the monitor mineral 

MMhb-2. These data, when combined with 

high-precision (≈ 0.2%) potassium 

concentration data from NIST, will enable 

the K/Ar age for MMhb-2 to be determined 

with a 10-fold improvement in accuracy. 

NIST is working with the U.S. 

Geological Survey to address 

the need for accurate standards 

to calibrate mass spectrometers 

at major argon-dating 

laboratories and thus resolve 

significant disagreements. 

Reference

Volume

939.685 cc 36Ar

38Ar in

Break-seal vials

40Ar

Argon Isotope Gas Supply

TM Pump

B.A.

Gauge

16.5 cc

Volume CDG

Pressure

Standards:

(3) 10 kPa RSGs

130 kPa RVG

1000 cc

40Ar:36Ar (30:1)

40Ar:36Ar (300:1)

B.A.

Gauge

T.M.

Pump

0.1 cc

0.3 cc

0.1 cc

U040

1000 cc 0.1 cc

U039

U041

U042

U046

Thermal

Pressure

Controller

0.1 cc

Atmospheric Argon

0.3 cc

U043

38Ar (~10-6mol)

38Ar (~10-6mol)

40Ar:36Ar (1:1)

1000 cc

3000 cc

3000 cc

3000 cc

N2

System used to calibrate 

the argon spike systems 
103

SEM images from a 16-element array in 

which TiO2 was grown by CVD with each 

element held at a different temperature. 

All images are at the same magnification.

Efficient Development of Materials for Chemical Sensing Using MEMS-Based Microarrays 

C.J. Taylor, D.C. Meier, and S. Semancik (836) 

Special types of thin film materials are required to perform various functions within chemical 

microsensors and microanalytical systems. Sensing materials, for example, must be tailored to 

detect the specific gases encountered in chosen application areas with sufficient sensitivity, 

selectivity, and speed. Other examples include preconcentrator and separator materials where 

chemical and microstructural properties must 

allow preferential adsorption or reaction at large 

number of surface sites. A combinatorial 

approach using MEMS-based microarrays has 

been developed to efficiently deposit and 

screen thin film sensing materials for detection 

of chemical warfare agents and planetary 

gases. 

Films of gradually varied composition and 

microstructure are deposited on an array by 

holding each element of the microarray at a 

different temperature. Microarrays of 4, 16 

and 36 thermally isolated and individually 

addressable microhotplate elements are 

used. Each ≈ 100 μm x 100 μm element can 

rapidly measure and control temperature 

(20 °C to 500 °C) locally, and also monitor 

the electrical properties of deposited films 

using surface microelectrodes. The primary 

advantage of the approach is that a large 

number of different sensing films can be 

fabricated and screened 

for sensing properties in 

one experiment and in a 

very small area. We 

have demonstrated this 

approach for the CVD growth of TiO2 (from titanium nitrate - see figure - and titanium 

isopropoxide) and SnO2 (from tin nitrate) sensing films. The microarrays enable highly efficient 

database development to correlate film processing and sensing performance. 

The multi-sample studies on TiO2 and SnO2 have proven extremely valuable in 

identifying deposition protocols for 

sensing films that perform well in a 

Defense Threat Reduction Agency 

(DTRA) -sponsored project for chemical 

warfare agent detection, and in a NASAsponsored project for detecting trace 

gases in planetary environments. We 

have also examined the thermal 

processing of high-area porous SiO2 structures from 

silsesquioxane-block copolymer blends for fabricating 

microscale preconcentrators. 

CSTL scientists have demonstrated 

that combinatorial microarray 

platforms can be used to accelerate 

the development of chemically 

functional materials for chemical 

sensing. The MEMS-based 

platforms are expected to be 

broadly applicable to other 

materials science research 

problems. 

The chemical sensing properties of 

metal oxide materials were explored 

using combinatorial methods for 

characterizing sensitivity, selectivity, 

and stability in sensing applications. 
104

Error-Free Liquid Flow Diverters for Calibration Facilities 

T.T. Yeh, P.I. Espina (836); and N.P. Yende (Council of Scientific and Industrial Research, 

South Africa) 

As NIST has refurbished its water flow rate measurement facilities, research on methods to 

reduce this gravimetrically based systemís measurement uncertainty has been performed. 

Diversion of the flow passing through metering devices into 

the systemís collection tank is an operation critical in 

accurate measurement of the flow rate. Various techniques 

have been suggested to reduce diverter valve errors in 

commonly used valve structures. A new concept has been 

developed that makes use of a double-action flow diversion 

process whereby the components of the diverter valve error 

are self-canceling. This self-cancellation effect occurs when the flow accumulation for the first and 

second diverter valve sweeps are functional inversions, summing to the full calibration flow at 

corresponding times in their diversion period. Several variations in the general approach have 

been developed conceptually and one of these options has been chosen for testing and 

implementation. Initial testing indicates 

substantial reduction in diverter error 

component contribution. A complete 

evaluation of this approach in ≈ 50 mm pipe 

sizes to quantify uncertainty component 

contributions is planned. If reduction in 

component uncertainty is significant, this 

design will be incorporated into the 

refurbishment of the main water flow rate 

measurement facilities at NIST. 

Hydrogen Profiling in Bulk Samples via Nuclear Analytical Techniques 

H.H. Chen-Mayer, D.F.R. Mildner, G. Lamaze, and R.M. Lindstrom (839) 

The ability to map hydrogen is a much sought-after goal in the field of non-destructive testing. 

Currently, many industrial and defense agencies, such as aircraft and auto industries, are highly 

active in imaging of components for inspection and reverse engineering purposes. Transmission 

neutron radiography has been the 

standard technique for imaging hydrogen 

in bulk samples, owing to the high 

penetration of neutrons in most 

materials. The Nuclear Methods Group 

is exploring various techniques for 

hydrogen profiling in bulk samples by 

combing prompt gamma activation 

analysis (PGAA) and neutron incoherent 

scattering (NIS) techniques, building on 

the established PGAA technique for 

NIST is improving national 

liquid flow measurement 

standards and methods to 

support improved accuracy 

in their dissemination. 

Reduced measurement uncertainty 

in liquid flows provides U.S. 

industry with improved accuracies 

and maintains U.S. leadership in 

flow rate metrology globally. 

Hydrogen in metals presents a significant 

challenge both scientifically and 

economically. As the simplest impurity, even 

at μg/g (ppm) levels it can cause structural 

degradation – a serious technical problem 

that still needs urgent solution after more 

than fifty years. On the other hand, for 

hydrogen storage, materials that allow 

highest hydrogen incorporation are desired. 

Exit flow

Rotating Flow 

divider

Translating

collecting 

channels

B: bypass loop

C: weighing tank 

B C B

Open sweep repositioning direct plate Close sweep 1

B C B

2

B C B

3

B C B

4

B C B

5

B C B

6

B C B

Exit flow

Rotating Flow 

divider

Translating

collecting 

channels

B: bypass loop

C: weighing tank 

B C B

Exit flow

Rotating Flow 

divider

Translating

collecting 

channels

B: bypass loop

C: weighing tank 

B C B

Open sweep repositioning direct plate Close sweep 1

B C B

2

B C B

3

B C B

4

B C B

5

B C B

6

B C B

Open sweep repositioning direct plate Close sweep 1

B C B

2

B C B

3

B C B

4

B C B

5

B C B

6

B C B

A rotating diverter valve with 

translating flow-directing channels
105

characterizing hydrogen standards prepared by volumetric hydrogen gas reaction with titanium 

metal. 

As a proof of principle, we simulate varying concentration of hydrogen in the matrix by using 

polypropylene films of 7 μm thick sandwiched in a stack of 1-mm thick titanium plates. The 

scattered intensity from the concentrated hydrogen in the films is greater than that from the 

titanium plates, resulting in vertical ridges in an otherwise smooth image viewed through a 0.5 

mm slit using a neutron-imaging detector. We can detect the change in the number of 

polypropylene films, and the change from a hydrogen-free titanium plate to one containing small 

amounts of hydrogen. We have established 

the relationship between the image intensity 

and the hydrogen concentration. Qualitatively 

these results demonstrate that the hydrogencontaining samples can be observed through a 

pinhole camera using NIS. Quantitative results 

have been obtained by image analysis that 

converts scattered intensity to the amount of 

hydrogen along the beam path, thereby 

achieving hydrogen profiling at a sub-mm scale 

and at 100 μg/g (ppm) levels. PGAA has been 

used to verify the absolute concentration of H, 

whereas NIS provides rapid and position 

sensitive H detection. 

Improved Vacuum Transfer Standards 

R.F. Chang (836) 

CSTLís goal is to disseminate NISTís realization of the Pascal at high vacuum levels directly to its 

customers and into widely used commercial gauges. However, commercial vacuum transfer 

gauges are unstable and hamper efforts to effectively disseminate NISTís uncertainties to its 

customers. In FY02, we identified the causes of instabilities in spinning rotor gauges (SRGs), 

which are one of the more stable type of vacuum transfer gauges. We quantified the effect of 

several factors on the accommodation coefficient (σ) of SRGs, which is directly tied to gauge 

calibration. These factors were selected because they typify the treatment of gauges and rotors 

of many calibration customers after they leave NIST. 

De-mounting and re-mounting of the 

suspension head caused a 0.1% shift in σ, 

which was comparable to the difference 

between baked and unbaked systems. 

Surprisingly, sudden loss of rotor suspension 

The NIS technique provides hydrogen 

information that is difficult to obtain by other 

methods. Compared to transmission 

radiography, scattering imaging has the 

advantage of greater contrast and lower 

noise. In addition, traditional neutron 

radiography suffers from the limitation that 

the component must be inserted between the 

beam and the imaging detector. In an NIS 

imaging system the detector can be placed 

on any convenient location, offering 

maximum flexibility for fieldwork when used 

in conjunction with portable neutron sources. 

The spinning rotor gauge (SRG) has 

become the transfer standard of choice 

for vacuum calibrations from 10-4Pa to 

1 Pa due to good calibration stability 

(changes < 0.5%/year). While internal 

NIST SRGs exhibit even better longterm stability, some gauges of 

calibration customers have shown 

inexplicably large shifts. 

Three SRGs show an average of ≈ 0.1% (0.4% 

max.) shift in calibration stability between 11 

successive mountings and de-mountings. 
106

Box Plot Comparison of Enthalpies of Fusion

for Indium from Adiabatic Calorimetry

NIST (2002)

LGC (1993)

NRC (1985)

NPL 1 (1979)NPL 3 (1979)

Oslo (1978)

Enthalpy of fusion / (J g-1) 28.1

28.2

28.3

28.4

28.5

28.6

28.7

did not have any measurable effect on σ, contrary to long-held belief. This work quantified the 

effects of several factors for the first time, but even taken in total, they cannot explain some of the 

larger instabilities observed in customer gauges. Additional aspects to explore include effects of 

magnetic polarization and other surface treatments upon the calibration stability of SRGs. This 

information will likely explain the remaining large calibration shifts observed by some customers, 

and also provide more data to help investigate the feasibility of an on-demand SRG calibration 

service. 

International Standards for Calorimetry and Thermodynamics 

D.R. Kirklin and D.G. Archer (838) 

Thermodynamic and calorimetric measurements have wide usage, ranging from product 

specification and quality control to research. Many calorimetric or thermodynamic instruments 

must be calibrated and methods validated for proper 

operation. A high-temperature adiabatic calorimeter 

has been made operational this year. This new 

instrument was used to measure enthalpies and 

temperatures of fusion of two certified reference 

materials (indium and tin) from the PhysikalischTechnische Bundesanstalt (PTB) for differential 

scanning calorimetry. The collaborative project with 

PTB was conducted under the Memorandum of 

Understanding signed between NIST, PTB, and 

Bundesanstalt f ̧r Materialforschung und -pr ̧fung 

(BAM). The newly determined enthalpy of fusion value for indium has an uncertainty that is about 

an order of magnitude better than that for the best 

previous measurements (see box 

plot). This new calorimeter will be 

used for certification of the 

temperatures and enthalpies of 

fusion of two more SRMs for calibration 

and validation of differential scanning 

calorimetry (DSC). 

New and better measurements for the enthalpy of 

combustion of SRM 217d iso-octane were undertaken 

this year so as to provide reference data for precision 

and bias statements required by some organizations 

such as ASTM. Certifications of SRM 2233, 

cyclopentane for calibration of DSC, and SRM 2234, 

gallium for calibration of DSC, were completed this 

year. These two SRMs are particularly important because low-temperature calibration of DSC must be accomplished 

with organic materials, which are not 

generally available in sufficient purity to 

be used in concert with literature 

values of temperatures and enthalpies 

of phase changes. 

Quantitation of these effects on SRGs has not been documented before, and in fact, goes 

against commonly held beliefs about their performance in the mid-range vacuum 

community. The results can significantly affect the treatment and operation of all SRGs, 

and will provide the basis for developing an uncertainty budget for the proposed ondemand SRG rotor calibration service.

Use of calorimetric and thermodynamic measurements ranges 

from product specification and 

quality control of manufacturing to 

state-of-the-art research. In 

response to these needs, NIST 

provides at least 14 current Standard Reference Materials (SRMs) 

for calorimetry. 

These accomplishments will allow NIST 

customers to perform more accurate 

calibrations of instrumentation and 

perform validation determinations of 

measurement methods. 
107

Laying the Foundation of Tandem Mass Spectrometry (MS/MS) and Ion Trap Libraries 

J.K. Klassen, Q.-L. Pu, and S.E. Stein (838) 

A program has been initiated to extend the coverage of the widely used NIST Mass Spectral 

Library to MS/MS and ion trap mass spectra. This has involved the creation and evaluation of an 

MS/MS library as well as the first NIST measurements of these spectra for inclusion in the library. 

A diverse MS/MS collection of 1,000 

spectra has been donated for use in our 

program by a major instrument vendor. 

One particular application area, pesticide 

analysis, has been targeted for initial 

development, and 600 spectra of 75 

compounds have been examined on ion 

trap instruments over a range of collision 

energies. We are now actively engaged in 

a round-robin exercise involving these 

pesticides with a number of environmental 

laboratories. A variety of measurements 

on a newly acquired triple quadrupole instrument have been 

made for both common calibration and internal standards and 

simple peptides. 

An important finding is that while the ion collision energy needs to 

be considered and controlled, variations in spectra between instruments are not too great to 

prohibit effective library searching. Also, a collection of ion trap spectra has been developed that 

includes specific varieties of compounds whose spectra are known to be different in ion traps 

than in conventional mass spectrometers. This will enable users of ion traps to be able to employ 

the NIST Library with fewer ëcorrectionsí for ion trap behavior. Approximately 200 such spectra 

have been measured in two ion trap instruments. This work has been done in close cooperation 

with the two ion trap instrument manufacturers. The analyzed substances are primarily relatively 

small organic molecules with low ionization energies and/or high proton affinities. The work has 

also led to improvements in the automated extraction of mass spectra by the NIST AMDIS 

program. 

The existing NIST mass spectral library is 

composed entirely of ‘classical’ electron 

ionization mass spectra. Spectra generated 

by ion trap and MS/MS instruments, which 

are becoming widely used for chemical 

identification, may not match these 

classical spectra. Additions to the NIST 

library are being made to enable its use 

with these two varieties of instruments. 

While ion trap and MS/MS spectra are routinely generated in 

laboratories in a wide range of application areas, reliable 

reference libraries are unavailable. With the addition of a 

limited number of carefully selected spectra to the current 

library, a major increase in the reliability of library searching is 

expected to result, especially for laboratories that are involved 

in the identification of small organic compounds. 

Future Plans: The ion trap library will be tested with blind or unknown GC/MS data to ensure that 

the database is large enough to improve identification of ion trap spectra. An MS/MS round-robin 

study with selected pesticides and conditions will be undertaken to further establish limits of 

variability in these spectra. Available ion trap and MS/MS spectra will be made available to 

current NIST library users. 
108

Molecularly Imprinted Stationary Phases for Microfluidic Chemical Separations 

A.C. Henry and L.E. Locascio (839) 

The synthesis of molecular imprint polymer (MIP) stationary phases that are specific for one type 

of molecule is being studied as part of an effort to develop microfluidic separation techniques. 

Work has focused on the creation of open tubular (as opposed to packed column) stationary 

phases that are covalently tethered to 

plastic microfluidic channels. The 

synthesis of the MIP films incorporates 

surface-bound alkene groups into the 

polymerization of methacrylic acid (MAA) 

in the presence of ethylene glycol 

dimethacrylate (EGDMA). Prior to the 

polymerization, a template compound is 

added to the mixture. The functional 

groups on the template molecule can interact by hydrogen bonding, etc. with the functional 

groups on the MAA. After polymerization, the template molecules are extracted, leaving behind 

ìpocketsî that are specific in size and shape to 

the template molecule. Two fluorophores with 

similar structures were used as template 

molecules for the fabrication of MIP thin films. 

Three films were synthesized: (1) an MIP film 

that employed mixed isomers of 

carboxyfluorescein as the template molecules, 

(2) an MIP film that employed 2í, 7í 

difluorofluorescein (Oregon Green) as the 

template molecule, and (3) a film that contained 

no template molecules. The template 

molecules were extracted into pH 9 carbonate 

buffer, and the three films were exposed to 1 

nmol/L Oregon Green in pH 7 phosphate buffer 

for a fixed amount of time and then rinsed. The 

films were then extracted over time in pH 9 

carbonate buffer and analyzed by fluorescence 

spectroscopy. As shown in the figure, the 

carboxyfluorescein-imprinted thin film seems 

slightly more specific for the Oregon Green molecule. This may be attributed to the structural 

similarities of the two molecules. When the films were exposed to Rhodamine B and then 

analyzed, no concentration trend was evident, implying only nonspecific surface interactions. The 

ultimate goal of this project is to 

synthesize MIP stationary phases 

tethered to the plastic microchannel that 

will allow for the separation of proteins 

on the basis of their quaternary and 

tertiary structures. In addition, this work 

may also lead to a benchtop separation 

system that will discriminate between 

racemates such as those regularly 

synthesized in the pharmaceutical 

industry. 

01234

0.00E+000

1.00E-011

2.00E-011

3.00E-011

4.00E-011

5.00E-011

6.00E-011

Concentration of Extract Solution (M)

Time Exposed to Extract Solution (h)

 control film

 MIP film imprinted for Oregon Green (1 nm Oregon Green exposure)

 MIP film imprinted for Carboxyfluorescein (1 nm Oregon Green exposure)

 MIP film imprinted for Oregon Green (water exposure)

 MIP film imprinted for Carboxyfluorescein (water exposure)

Extraction of Oregon Green from MIP 

matrix after exposure to 1 nmol/L Oregon 

Green in pH 7 phosphate buffer 

Molecular imprinted polymer (MIP) stationary 

phases that are specific for a certain type of 

molecule may lead to separation techniques 

with enhanced selectivity. Potential 

applications include chiral molecules of 

interest to the pharmaceutical industry. 

CSTL researchers are developing MIP stationary 

phases that can be used in capillary electrochromatography separations of similar molecules. 

This work could significantly impact 

characterization of chiral pharmaceuticals 

because the tools being developed would be costeffective, small, and require only minimal amounts 

of solvents, making them appropriate for use in 

high-throughput screening. 
109

SRMs for Near Infrared (NIR) and Raman Spectroscopy 

W.S. Hurst and J.E. Maslar (836); E.S. Etz (837); and S.J. Choquette (839) 

Raman spectroscopy is now finding its place in the industrial environment for process 

measurements and quality control. The lack of accepted practices, standards and spectral 

libraries has been a main obstacle to the acceptance of Raman in industrial settings and is a 

barrier to its use in regulated industries. At the present time, Raman spectra are instrument 

dependent and a convolution of the spectrometer instrument response and scattering 

characteristics of the sample. The series of 

SRM 224x standards is intended to largely 

solve this problem. These are glasses that 

luminesce when irradiated 

with the spectrometersí 

laser. The shape of this 

luminescence is certified 

by NIST and is described 

by a simple polynomial expression. This polynomial, together with a 

measurement of the luminescence spectrum of the SRM, can be used to 

determine the spectral response correction that is unique to 

each instrument. This will enable the correction of a sample 

Raman spectrum so that it is system independent. SRM 2241 

Relative Intensity Correction Standard for Raman 

Spectroscopy for systems operating with 785 nm excitation 

was certified this year. SRM 2036 is a newly introduced NIR 

diffuse reflectance wavelength standard. This standard 

provides the same level of calibration confidence as NISTís 

existing SRM 1920a but is easier to produce, utilizes the 

same materials as SRM 2036/2065, and 

eliminates the strong dependence of band 

position on the resolution of the instrument that is 

inherent in the use of SRM 1920a. These new 

standards enable the calibration of these 

spectrometers at a fraction of the cost of current 

methods and resolve several technical issues 

with an existing SRM. The use of these 

standards will enable the development of 

standardized Raman spectral libraries, 

instrumentñto-instrument comparisons, and 

transfer of calibration methods between labs and instruments. 

Critical Evaluation of XRF for Making Matrix-Independent Quantitative Chemical 

Measurements 

J.R. Sieber and A.F. Marlow (839) 

Research in matrix-independent Xray fluorescence spectroscopy 

(XRF) methods has progressed 

apace of metal SRM development 

projects. Building on the foundation 

laid during Portland cement and 

zeolite SRM development work and 

the efforts of scientists at 

Corporation Scientifique Claisse, 

techniques were developed to prepare metals and alloys for borate fusion. Once the metal (chips 

The introduction of SRM 2241 and the next 

set of glasses for 532 nm, 488nm to 514 nm 

and 1064 nm Raman excitation sources will 

enable the use of this technique in 

industries that are regulated by the FDA. 

The lack of performance validation and 

system correction standards has inhibited 

this technique from being widely used in 

the pharmaceutical, chemical, and 

forensics community. 

Users of Raman and NIR instrumentation 

in the pharmaceutical community and 

other industries need standards that 

provide traceability. CSTL scientists are 

addressing this need through the 

introduction of two new SRMs. 

Matrix-independent methods of analysis, regardless 

of the instrumentation, offer direct traceability to 

the mole through calibration using ultrahigh purity 

materials and gravimetric preparation. This is 

critically important to the global metrology 

infrastructure. Such methods can provide the same 

benefits to industry labs thereby reducing the 

practice of calibrating with SRMs. 
110

or powder) is oxidized, it can be fused and synthetic standards can be prepared for a matrixindependent calibration. Relative expanded uncertainty estimates as low as 0.2 % have been 

achieved for major and minor constituents of steel and aerospace alloys. 

Low uncertainty and calibration using high-purity compounds makes the 

results directly traceable to the mole. 

New Version of the NIST Electron Elastic-Scattering Cross-Section Database 

C.J. Powell (837); A. Jablonski (Polish Institute of Physical Chemistry); and F. Salvat (Univ. 

of Barcelona) 

Cross sections for the elastic scattering of electrons by atoms are needed for modeling the 

transport of electrons in materials by Monte Carlo simulations and other means. Such transport 

calculations are needed to improve the accuracy of surface analyses, bulk analyses, and thin-film 

analyses of inhomogeneous samples by the techniques of Auger-electron spectroscopy (AES), Xray photoelectron spectroscopy (XPS), electronprobe microanalysis (EPMA), and analytical 

electron microscopy (AEM). The cross sections 

are also needed for similar transport calculations 

in other applications including radiation dosimetry, 

radiation therapy, radiation processing, radiation 

protection, and electron-beam lithography. 

A new version of the NIST Electron Elastic-Scattering 

Cross Section Database (SRD 64) was completed in 

FY2002 and is expected to be released in early FY2003. 

This version (Version 3.0) contains two major changes 

from Version 2.0. First, the cross sections and related 

data were calculated from a relativistic Dirac partialwave analysis in which the potentials were obtained 

from Dirac-Hartree-Fock electron densities computed 

self-consistently. This potential is believed to be more 

reliable than the Thomas-Fermi-Dirac potential used 

previously. Second, the upper energy limit for the 

cross-section data has been extended from 20 keV to 

300 keV. As a result, the database should be useful 

for a wider range of materials-characterization 

applications. 

Odd/Even Chain Length Effects on Solute Retention in Reversed-Phase Liquid 

Chromatography 

L.C. Sander and C.A. Rimmer (839) 

Chromatographic separation of organic compounds in complex sample matrixes is often a 

requisite to the accurate and reproducible 

measurement of targeted species for applications 

such as healthcare, food and nutrition, and the 

environment. Although liquid chromatography 

(LC) is a mature analytical technique, separations 

are often developed from empirical observation 

rather than theory, and solute retention remains a 

topic of active consideration. As part of an 

ongoing effort to understand fundamental chromatographic processes in LC, a series of columns 

were prepared to probe relationships between solute retention and stationary phase properties. 

This reference data should make it 

possible to make more accurate 

simulations of electron transport to 

provide more reliable analyses of 

inhomogeneous specimens by AES, 

XPS, EPMA, and AEM. There will be 

similar benefits for other applications 

such as radiation dosimetry and 

electron-beam lithography. 

Reference data is needed to improve 

the accuracy of surface analyses by 

Auger-electron spectroscopy and X-ray 

photoelectron spectroscopy and for 

bulk and thin-film analyses by electronprobe microanalysis and analytical 

electron microscopy. 

CSTL scientists are studying 

fundamental processes in liquid 

chromatography (LC), with the goal of 

improved analytical methods for 

applications in healthcare, food and 

nutrition, and the environment. 
111

Stationary phases were synthesized with alkyl chain 

lengths ranging from 13 to 18 carbons, by three 

different synthetic approaches (monomeric, 

polymeric, and self-assembled monolayer). In 

addition to the effect of chain length, the influence of 

temperature, bonding density, and bonding chemistry 

on chromatographic performance was evaluated by 

use of SRM 869a Column Selectivity Test Mixture for 

Liquid Chromatography. 

The phases were characterized in terms of 

methylene selectivity, shape selectivity, and band 

broadening. No significant chromatographic 

differences were found to result from the synthetic 

routes, except that the different bonding chemistries 

provide a different range of bonding densities. The 

approach used in bonded phase synthesis limits the 

density of the alkyl chains that can be achieved. For 

bonding densities ranging from 2 μmol/m2

 to 8 μmol/m2, a linear increase in methylene selectivity 

was observed with increasing bonding density. Longer chain length columns with higher bonding 

densities provided enhanced selectivity for 

shape-constrained solutes. These findings may 

facilitate the development of improved analytical 

methods for classes of analytes such as 

vitamins (carotenoids, retinoids, tocopherols), 

biomolecules (steroids) and environmental 

contaminants (polycyclic aromatic hydrocarbons, 

polychlorinated biphenyl congeners). 

Assessment of Pressurized Fluid Extraction for Enhanced Extraction of Environmental 

Samples 

M.M. Schantz, B.A. Benner, Jr., D.L. Poster, and S.A. Wise (839) 

Until recently, Soxhlet extraction with a variety of solvents was the only extraction method 

employed for certification measurements of organic constituents in natural environmental-matrix 

SRMs such as sediment, tissue, and particulate matter. The typical Soxhlet extraction requires 

16 h to 20 h of extraction time using 200 mL to 300 mL of solvent. With the demand for reduced 

solvent usage and faster analyses, alternative extraction techniques have been investigated. 

Extraction methods such as supercritical fluid extraction (SFE) and pressurized fluid extraction 

(PFE) have been evaluated and 

compared to traditional Soxhlet 

extraction for the determination of 

organic contaminants such as 

polychlorinated biphenyls (PCBs), 

chlorinated pesticides, and polycyclic 

aromatic hydrocarbons (PAHs) in environmental-matrix SRMs. SFE typically uses carbon dioxide 

with the addition of more polar solvents such as methanol as modifiers; PFE uses conventional 

liquid solvents at elevated temperatures and pressures (100  ̊C to 200  ̊C and 7 MPa to 14 MPa). 

The purpose of these investigations was to demonstrate whether SFE and PFE were comparable 

to Soxhlet extraction for the determination of organic contaminants in natural environmental 

matrices and as such could they be used in the certification of such natural-matrix SRMs. 

Traditional Soxhlet extraction is time-consuming 

and requires large volumes of solvents. CSTL 

scientists are investigating alternative extraction 

methods for natural-matrix SRMs. 

This work has resulted in an improved 

understanding of solute retention 

mechanisms in LC which will facilitate the 

development of improved analytical 

methods based on knowledge of column 

properties (rather than a trial and error 

approach). Ultimately, measurement 

precision and accuracy may improve for 

relevant analytical problems. 
112

0

1

2

3

4

5

6

micrograms/gra

m

I[1,2,3-cd]P

B[ghi]P

DB[a,h+a,c]A

Picene

B[b]C

Certificate (1985)

Soxhlet (CH2Cl2)

PFE (CH2Cl2)

PFE (Tol:MeOH)

Comparison of Soxhlet extraction and 

PFE for extraction of higher molecular 

weight PAHs in SRM 1650 Diesel 

Particulate Matter 

SFE was found to be matrix 

dependent for the extraction of 

PCBs and PAHs with comparable 

extraction efficiencies for PCBs 

and PAHs in sediment but 

significantly lower efficiencies for 

higher molecular weight PAHs (> 

MW 252) in air and diesel 

particulate matter. In evaluating 

the applicability of PFE for use as 

an alternative method of solvent 

extraction during the certification 

of natural environmental-matrix 

SRMs, the temperature and 

pressure conditions were 

optimized for the extraction of 

PCBs, pesticides, and PAHs from 

sediment, fish and mussel tissue, 

and air and diesel particulate 

matter. For these matrices, the comparability of PFE to Soxhlet extraction for the determination 

of PAHs, PCBs, and chlorinated pesticides was demonstrated. For the diesel materials, PFE 

showed greater extraction efficiencies by 

about 20% than Soxhlet extraction for the 

higher molecular weight (MW) PAHs, such 

as indeno[1,2,3-cd]pyrene (MW 276), 

benzo[ghi]perylene (MW 276), dibenz[a,h + 

a,c]anthracene (MW 278), picene (MW 278), 

and benzo[b]chrysene (MW 278), as shown 

in the figure for SRM 1650 (Diesel 

Particulate Matter). 

PFE has also been demonstrated to be useful for the extraction of microsamples (as small as 

several mg), which allows for the determination of the microhomogeneity of these natural-matrix 

SRMs. 

Ruggedness Testing of HP-ICP-OES for Single and Multielement Determinations 

M.L. Salit and G.C. Turk (839) 

A high-performance approach to the measurement of elemental composition using inductively 

coupled plasma optical emission spectroscopy was developed at NIST several years ago in order 

to make detailed comparisons of elemental calibration solutions. This technique uses an 

innovative experiment design coupled with 

a novel drift correction approach to 

minimize and quantify the major sources 

of uncertainty in this chemical analysis. 

The performance of this method, HighPerformance Inductively Coupled Plasma 

Optical Emission Spectrometry (HP-ICPOES) was recently documented for 64 

elements, where the typical relative expanded uncertainty observed is 0.1% (Analytical Chemistry 

2001, 73, 4821-4829). This technique utilizes unmodified, commercially available equipment, is 

well suited to automation, and yields results comparable to the best attainable by so-called 

PFE has now been incorporated into the 

analytical measurement scheme for the 

certification of environmental-matrix SRMs and 

has been used to assess the microhomogeneity 

of these SRMs. PFE is now being evaluated for 

use in the extraction and measurement of active 

and/or marker organic constituents in botanical 

dietary supplements. 

CSTL researchers are expanding the 

applicability of HP-ICP-OES to measurement 

problems in optoelectronics and high-purity 

metals, and transferring this measurement 

capability to laboratories outside of NIST, 

including precious metal refiners and 

commercial standards producers. 
113

According to Metalor “ the NIST-developed HP-ICPOES technique has revolutionized 

our Platinum Group Metal (PGM) assay lab. Assay completion times that ran 20 to 30 

days now average 4 to 6 days. Assay precisions have improved by more than an 

order of magnitude. Accuracy has greatly improved...” 

ìclassicalî methods, the craft- and labor-intensive benchmarks of best practice. HP-ICP-OES is 

now being expanded beyond the original intended application to multielement analyses and 

complex matrix samples. In the past year the technique has been successfully applied to the 

analysis of compound semiconductors for optoelectronics, high-purity metals, and platinum group 

metals both at NIST and at private-sector laboratories. We have worked closely with laboratories 

wishing to duplicate our capabilities, and have 

developed software tools to enable this 

technology transfer. Using their existing 

instrumentation, these laboratories have seen 

order-of-magnitude reductions in their 

measurement uncertainties, with dramatic 

impact on their laboratory operations. 

SpectroML—an Extensible Markup Language for Molecular Spectroscopy Data 

A. Arslan, A.D. Nguyen, J.C. Travis, R. Schäfer, and G.W. Kramer (839) 

The interchange and storage of analytical chemistry data has long been hampered by multiple, 

incompatible data formats. However, in the last few years, the emergence of platform and 

application independence through the 

Internet has advanced the possibilities for 

data interchange beyond the capabilities of 

these protocols. Todayís analytical 

chemists need not only to interchange data 

generated within a specific technique, they 

need to interchange, import, export, store, 

and combine all their data from multiple 

sources at multiple sites. Users are now no longer content with just having ìpicturesî of their data 

(i.e., data plots). They need real access to their data from within a report itselfóthey 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. 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. 

We have created an XML-based markup language for UV/visible data called SpectroML and are 

applying it to the interchange of data from our spectrometers and our optical filters database. 

SpectroML was created initially to serve as a model implementation of a markup language for 

molecular spectrometry and ultimately to provide a web-based mechanism for interchanging 

UV/visible spectral data generated on different spectrophotometers among ourselves, our 

colleagues at other national metrology institutes, and our NIST-Traceable Reference Material 

(NTRM) vendors. We have worked with the ASTM E13 Molecular Spectrometry and 

Chromatography Committee and representatives from a number of instrument companies to 

create a task group for developing a unified XML-based approach for interchanging molecular 

spectrometry and chromatography data. This year we completed the software to convert the data 

Multiple, incompatible data formats have 

hindered the interchange and storage of 

analytical chemistry data. An XML-based 

markup language is being developed by 

NIST scientists to facilitate data exchange. 

The implementation of HP-ICP-OES is only 

in its infancy, but comments received from 

collaborators outside of NIST indicate that 

the implementation of HP-ICP-OES has had 

a very favorable impact on their operations.
114

files from our reference spectrometer (HAS-II) to SpectroML and are currently working to do the 

same for the Perkin Elmer Lambda 900 instrument. We have recently begun the task of 

interfacing SpectroML to our optical filters database. With SpectroML as the lingua franca, we 

will need only one application to interface all our instruments to the database. When this software 

is in place, the task of quality assurance monitoring for our NTRM optical filters program will be 

automatable. 

Transducer-Assisted Piston Gauge Calibrations 

D.A. Olson (836) and T. Kobata (National Metrology Institute of Japan) 

Existing piston gauge calibration methods require a great deal of operator 

skill and experience to find a pressure balance point and render a decision 

about when the equilibrium is ìsufficientî so that the next datum can be set 

and acquired. Our objective is to improve piston gauge calibrations by 

reducing or removing 

operator judgment and manual data entry, and 

introduce more consistency through 

automation, with no penalty in overall 

uncertainty. To meet this objective, a method 

to automate the data-gathering process in the 

calibration of one piston gauge against another 

has been developed. Rather than adjusting 

small ìtrimî masses until the 

balances generate equal pressure, 

the two balances are brought into 

approximate pressure equilibrium 

and a pressure transducer 

measures the remaining pressure 

difference. The transducer-assisted 

traditional fall-rate methods have 

been demonstrated to agree with 

the combined standard uncertainty 

of the methods in nearly all cases, 

and to within the combined 

expanded uncertainty (k=2) for all 

cases. The combined standard 

uncertainties for this method are 53 

Pa to 112 Pa, making them on the 

same order as the resolution of the 

pressure transducer (56 Pa). The 

relative combined standard uncertainties are 3.6x10-6 or less at 20 MPa, and 1.1x10-6 or less 

above 50 MPa, and compare quite favorably with the relative combined standard uncertainty of 

the NIST Transfer Standard at 280 MPa, which is 16x10-6 (k=1). To keep the uncertainty in the 

method comparable to the uncertainty of the fall rate method, the pressure difference between 

the two piston gauges, in this case, was |∆P| ≤ 105

 Pa, which keeps the transducer uncertainty 

equal to or less than other component uncertainties. An unexpected advantage of the transducerassisted method is far greater sensitivity over extant methods, which also allows us to observe 

Schematic of hardware set-up for 

Transducer Assisted Calibration 

The development of an XML-based universal molecular spectrometry and chromatography 

markup language could 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. 

NIST is improving its calibration services 

through the development of a new 

system for pressure gauge calibration. 

This system reduces the dependence on 

operator judgment and provides more 

consistency through automation. 
115

The transducer-assisted method has already 

impacted NIST calibration services and allowed us 

to discover systematic problems in a recently 

purchased piston gauge that could not have been 

noticed using traditional calibration methods. 

Widespread adoption of this straightforward 

method will provide piston gauge manufacturers a 

far more sensitive diagnostic tool than ever before 

available, and could culminate in substantive 

performance improvements for this mature field. 

2nd-order effects such as effective area 

dependence on piston stroke height. 

Thus, we have achieved our objective to 

improve piston gauge calibrations and 

introduce more consistency through 

automation, with no penalty in overall 

uncertainty. 
116
117

11. Microelectronics 

CSTL supports the microelectronics 

industry by providing the necessary 

physical standards for process 

control of parameters such as 

temperature and humidity. CSTL 

also provides reference materials to 

facilitate quality control in manufacturing, as well as providing 

reliable kinetic and thermophysical property data to facilitate 

process modeling, to meet critical industry needs. CSTL research remains on the cutting edge in 

order to provide dramatically improved measurement tools to keep advanced U.S. 

microelectronics manufacturing globally competitive. 

Completion of Suite of Dopants in Silicon Standards 

R.L. Paul, R.R. Greenberg, R.M. Lindstrom (839); and D.S. Simons (837) 

The U.S. semiconductor industry relies heavily on secondary ion mass spectrometry (SIMS) for 

characterization of the depth distribution of critical dopants (boron, arsenic, and phosphorus) in 

silicon. To achieve high accuracy in the concentration determination by SIMS, standards of 

known dopant concentration, conveniently provided by ion 

implants of certified dose, are required. International 

SEMATECH (a consortium of semiconductor manufacturers) 

recently listed SRMs of implanted phosphorus, boron, and 

arsenic in silicon as a high-priority need. Standard 

Reference Materials of boron and arsenic implants in silicon 

(SRMs 2137 and 2134) have previously been developed by 

NIST for use as SIMS calibration standards. In pursuit of a phosphorus in silicon SRM, a 

radiochemical neutron activation analysis (RNAA) procedure was developed, critically evaluated, 

and shown to have the necessary sensitivity, chemical specificity, matrix independence, and 

precision to certify phosphorus at ion implantation levels in silicon. A phosphorus-in-silicon SRM 

(SRM 2133) was prepared by implantation of a 200 mm 

diameter silicon wafer. The wafer was cut into 1 cm2

pieces for analysis by RNAA. All potential sources of 

uncertainty were evaluated in order to establish RNAA as 

a primary method for certification. The method was used 

to certify the phosphorus concentration of SRM 2133 

Phosphorus Implanted Silicon as 9.55 x 1014 atoms·cm-2 ± 

0.12 x 1014 atoms·cm-2. 

Development of Process Monitoring and Diagnostic Techniques 

M.A. Sobolewski, K.L. Steffens (836); and E.C. Benck (PL) 

Plasma processing reactors have historically been 

designed and operated using empirical methods alone, 

but continued evolution of these tools requires a much 

greater reliance on process and reactor modeling. 

Indeed, model-based process design and control is an 

important need identified in the National Technology 

Roadmap for Semiconductors. To obtain more reliable 

predictions of the spatial uniformity, chemistry, and 

electrical properties of processing plasmas, further 

progress in model development and validation is 

The three SRM implants in silicon 

will allow the semiconductor 

industry to achieve low 

systematic error for dopant 

concentration measurements as 

specified in the 2001 International 

Technology Roadmap for 

Semiconductors. 

Ion-implanted standards of 

known dose for B, As, and P 

are needed for accurate 

concentration-depth profiles 

of these dopants in silicon. 

Advanced measurement 

methods, data, and models are 

needed to characterize plasma 

etching and deposition 

processes important to the 

semiconductor industry, enabling 

continued progress in process 

optimization, process control, 

and model-based reactor design.

The U.S. electronic 

component industry 

has grown in recent 

years and shipments 

are expected to exceed 

$300 billion by 2004. 
118

Heterogeneity Test for Si, Ge, and SiGe Wafers 

Expanded Uncertainty (99%) mass fraction (in %) 

Sample SiK1 SiK5 GeL1 GeL5 

StdWafers 0.94% 1.36% 0.60%

SiGe14 1.22% 1.29% 1.38% 1.55%

SiGe6.5 1.75% 1.55% 4.67% 4.64%

required. Also, to enable improvements in process control, a need exists to develop sensors that 

are compatible with the manufacturing environment. 

In dielectric etching plasmas, CF is an important species to 

understand because it is thought to participate in the formation 

of the fluorocarbon polymer layer which enables selective 

etching of SiO2. Planar laser-induced fluorescence (PLIF) has 

been used to measure twodimensional density maps of CF 

as a function of power and 

pressure in CF4/O2 and C4F8

plasmas and as a function of power in CF4 with and without a Si 

wafer. Spatial variations in plasma temperature can cause spatial 

differences in gas density and reaction rates. Thus, understanding 

gas temperature in etching plasmas is also important. The 

technique of PLIF of the CF radical has been extended to enable 

two-dimensional temperature mapping in fluorocarbon plasmas. 

Temperature maps have been measured in CF4 plasmas from 27 

Pa (200 mTorr) to 107 Pa (800 mTorr). Temperature spatial 

variations of up to 150 K have been observed in these plasmas. 

Temperature increases with pressure and power is lowest near the 

cooled electrode surfaces. 

Development of SiGe Compositional Standards for the Semiconductor Industry 

R.B. Marinenko, J.T. Armstrong, S. Turner, E.B. Steel, and D.S. Simons (837) 

SiGe technology is having a profound impact on the 

wireless and computer industries enabling the 

development of electronic components that are smaller, 

faster, less noisy, require less power, and are nearly as 

inexpensive to produce as silicon components with little 

retooling. With this new technology has come the need 

to characterize the 

thin films used in 

the new devices. 

We are working with the semiconductor industry to use the 

electron probe microanalyzer (EPMA), Auger analysis, 

analytical electron microscopy (AEM), and secondary ion 

mass spectrometry (SIMS) to develop bulk and thin-film 

materials that can be used as compositional standards for 

SiGe thin film manufacturing. 

Bulk SiGe wafers cut from single-crystal boules were 

evaluated with the EPMA for micro- and macroheterogeneity for use as primary standards for 

future characterization of SiGe thin films on Si that are needed by the microelectronics industry as 

reference standards. Specimens 

with nominal atom fractions of 14 % 

Ge and 6.5 % Ge were rigorously 

tested with wavelength dispersive 

spectrometers (WDS) using multiple 

point, multiple sample, and duplicate 

data acquisitions. These data were 

compared to pure Si and Ge wafers 

tested under the same experimental 

Measurement techniques, 

data, and models provided by 

NIST assist industry in 

improving their plasma 

modeling and characterization 

efforts. 

Next-generation computer chips 

and most current cellular 

telephones are based on SiGe 

technology. Industry has 

requested a compositional 

standard to meet this growing 

industry usage. CSTL scientists are providing 

the semiconductor industry with 

a needed SiGe bulk reference 

material that is the first step in 

providing the thin-film reference 

material it needs for the 

manufacture of SiGe wireless 

and next-generation 

microprocessor components. 

Plasma processing 

reactor 
119

procedures. The SiGe14 had the least heterogeneity with an overall expanded uncertainty (99%) 

in the mass fraction of 1.6 % or less for both elements while the SiGe6.5 specimen had an 

expanded uncertainty in the mass fraction as large as 4.7 % for Ge and less than 1.8 % for Si. 

Critical Evaluation of GDOES for Depth Profiling of Layered Materials 

M.R. Winchester (839) 

While the quantitative determination of elemental 

composition as a function of depth is likely to become 

even more important in the future, this remains a difficult, 

time consuming, and expensive metrological problem. 

Consequently, there is a need for better, faster, and 

more economical characterization methods. At NIST, 

glow discharge optical emission spectrometry (GDOES) 

is being developed for this purpose. GDOES is 

inherently much faster and much less expensive than most competing methods of surface 

analysis. 

The capabilities of GDOES depth profiling are demonstrated through the development of a 

GDOES method for the characterization of SRM 2137 Boron Implant in Silicon Standard for 

Calibration of Concentration in a Depth Profile. This sample consists of a single crystal Si 

substrate into which 10B has 

been implanted at a nominal 

energy of 50 keV. The surface 

was rendered disordered by Si 

ion implanation prior to the 

implantation of 10B. A GDOES 

depth profile obtained for SRM 

2137 is shown in the figure. The 

corresponding Secondary Ion 

Mass Spectrometry (SIMS) 

depth profile is also shown for 

comparison. The GDOES data 

were acquired at glow discharge 

conditions of 1000 V / 30 mA 

using a sampling frequency of 5 

Hz. Five individual depth 

profiles were coadded, in order 

to improve the signal-to-noise 

ratio. A low-pass Fast Fourier Transform (FFT) was then applied to the coadded profile for 

smoothing. The GDOES concentration axis was not calibrated for this work, but was simply 

adjusted to allow the overall shape of the depth profile to be compared with the quantitative SIMS 

data. The GDOES depth axis was calibrated using independent measurements of the sputtering 

rate performed via profilometry. The results are encouraging, in that the GDOES depth profile 

matches very well previous depth profiles obtained 

using SIMS. This points out the potential usefulness of 

GDOES depth profiling. Once fully developed, GDOES 

should provide United States industry with a useful 

metrological tool, potentially affecting many industries 

that rely upon thin films, coatings, and other surface 

layers. 

CSTL researchers are working to 

improve the analytical capabilities 

of GDOES, especially with regard to 

analytical sensitivity, depth 

resolution, and the accuracies of 

the concentration and depth axes.

0.00E+00

1.00E+19

2.00E+19

3.00E+19

4.00E+19

5.00E+19

6.00E+19

7.00E+19

8.00E+19

9.00E+19

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Depth, micrometers

SIMS

GD-OES

The quantitative determination 

of elemental composition as a 

function of depth on surfaces 

is an important analytical 

problem in a wide variety of 

modern technologies. 
120

Critical Evaluation of NDP and PGAA for Determination of Boron in Silicon 

G. Lamaze, H.H. Chen-Mayer, R.M. Lindstrom, E.A. Mackey, and R.L. Paul (839) 

A critical evaluation of Neutron Depth Profiling (NDP) 

and Prompt Gamma Activation Analysis (PGAA) has 

been undertaken to determine the state of uncertainties 

of these methods with the goal of identifying significant 

sources of error that can be reduced in the future. 

Additionally, new values of the minimum detectable 

amounts of boron in silicon have been established. In 

NDP, experimental measurements of stopping powers have led to new values that are about 5% 

different from literature values, thereby reducing 

uncertainties from that source. The improved values of 

the stopping power lead to increased accuracy in the 

determination of film thickness and density by the NDP 

method. PGAA has established a new lower limit of 

detection for boron in silicon that is 3 ng/g. We reported 

complete uncertainty budgets at the 2002 Winter Meeting 

of the American Nuclear Society in Washington. The 

establishment of a complete uncertainty budget for both 

NDP and PGAA allows the method to be quantitatively 

compared with other analysis methods. Establishing 

minimum detection limits for boron in silicon allows customers to know 

immediately whether our techniques are applicable for their needs. Future 

work includes a new cold neutron beam line for both PGAA and NDP to be 

installed in the near future that will lower backgrounds significantly and should 

lead to both lower detection limits and lower uncertainties. This will also lead 

to a new NDP lower detection limit of boron of 5 x 1015 at/cm3

 (approximately 

40 ng/g in bulk samples). 

Far-Ultraviolet Spectra of Compounds Important in Next-Generation Lithography 

V.L. Orkin (838) 

Atmospheric pressure deep UV lithography using fast chemically amplified photoresists will be 

the mainstay of semiconductor production into the foreseeable future. The development of 

materials and techniques for both 193 nm (based on ArF excimer lasers) and 157 nm (based on 

F2 excimer lasers) exposure tools for UV lithography 

should soon become available. A challenge for the 

utilization of these shorter wavelengths is 

photodeposition on the surfaces of the many lenses 

required to focus and direct the light. These deposits 

cause light scattering and poorer imaging. Although the 

problem has been observed to some extent at longer 

wavelengths, it becomes very serious for the deep UV 

radiation. The control of this problem requires a 

quantitative understanding of the photochemical properties of the gases that are likely to be 

present in the lithography apparatus. To help address these problems, we have measured the 

deep UV spectra of a set of silicone-containing compounds likely to be used in next-generation 

lithography. 

Because nuclear methods are 

able to provide quantitation in a 

non-destructive measurement, 

they provide cornerstones for 

other measurement techniques. 

The establishment of a complete 

uncertainty budget for both NDP 

and PGAA allows the method to be 

quantitatively compared with other 

analysis methods. Establishing 

minimum detection limits for boron 

in silicon allows these techniques 

to fit into the semiconductor 

roadmap. 

Important contamination 

sources in lithography were 

chosen for study based on 

input from the first Lithography

Contamination Control and 

Monitoring Roadmap 

Workshop. 
121

The absorption spectra of nine different silicone-containing compounds were measured down to 

160 nm (the limit of our apparatus). With the exception of silane, all the Si-containing gases 

studied exhibit very strong absorption at shorter 

wavelengths. At 193 nm, the absorption cross 

sections are relatively small, except for 

hexamethyldisilazane. For hexamethyldisiloxane 

and the alkyl-methoxysilanes, the absorption 

cross section increases by more than two orders 

of magnitude between 193 nm and 160 nm. 

 

Measurements for Vacuum Process Control 

R.F. Berg (836) 

The semiconductor electronics industry uses thermal mass flow controllers (MFCs) to control 

processes such as plasma etching. A wide variety of toxic, flammable, and corrosive gases are 

used at flow rates below 1000 μmol/s (1 μmol/s ≈ 1.3 

standard cm3

/min.). This variety challenges the companies 

that manufacture and calibrate MFCs. 

A few years ago, participants at an MFC workshop at NIST 

predicted that industry requirements for flow uncertainty will 

fall below 1%, and they called for corresponding 

improvements of national standards. In response, we are developing and improving standards for 

gas flow in the range from 0.01μmol/s to 1000 μmol/s. We have developed a flow metering 

approach based on capillaries as a 

transfer standard having improved 

reproducibility. The capillary flow meter 

(CFM) is a second-generation transfer 

standard whose flow elements consist of 

long, coiled, quartz capillaries. A 

hydrodynamic model of the CFM standard 

has been developed and validated against 

two primary flow standards. Tests with 

nitrogen agreed with both primary 

standards at flow rates from 0.1 to 1000 

μmol/s. Tests with other gases verified 

that the model correctly handles gases 

with widely varying thermophysical 

properties. A comparison with Italy’s metrology institute (IMGC) showed agreement to within 0.1 

% from 0.2 μmol/s to 800 μmol/s. Flow comparisons with two U.S. manufacturers of gas flow 

NIST is working with the 

semiconductor industry to 

address the need for more 

accurate MFC performance. 

The results of this work provide the 

necessary background information 

that will allow plans for the design of 

next-generation lithography 

equipment to proceed with 

quantitative information on the 

photochemical behavior of this 

compound set. 

http://www.nist.gov/kinetics/spectra
122

standards tested each company’s primary standards 

to high accuracy and demonstrated NIST’s emerging 

capability to measure small gas flow rates with 

excellent precision and accuracy. 

Non-Contact Free Carrier Density Measurements for Compound Semiconductors 

J.E. Maslar and W.S. Hurst (836) 

Transport of free carriers is central to the operation of all optoelectronic devices and reliable 

measurement of the carrier properties is critical. Hall or capacitance-voltage measurements are 

traditionally used to obtain this information, but require 

electrical contact. This precludes their use in situ during 

growth or processing and, typically, even on actual device 

layers. Raman spectroscopy, as an optical technique that can 

be used for transport property determination, does not suffer 

from these limitations. In addition, it is non-destructive, 

spatially resolved, and can be applied to a specific buried 

layer, sometimes a problem for traditional 

electrical measurements. A number of issues 

exist that are central to determining the accuracy 

and precision of this method, including the 

semiconductor under investigation, measurement 

system parameters, and Raman spectral model 

used to fit the measured spectra. NIST is 

systematically addressing such issues. 

Raman spectroscopic systems, optimized for 

narrow and wide band gap materials, were 

assembled and used to measure the Raman 

spectra of n-type doped GaN, GaAs, and GaSb 

films. An example of the measured Raman 

spectrum for n-type GaSb is given in the figure. 

Fitting the data with a Raman spectral model 

results in a determination of carrier concentration 

of ≈ 6 x 1018 cm-3. Relatively simple spectral 

models were developed and employed to 

determine free carrier concentrations from the 

experimental Raman spectra. This project facilitates the utilization of Raman spectroscopy for 

spatially resolved off-line characterization, as well as process monitoring and control, during film 

growth and etch processing. A potential user should be able to determine the expected accuracy 

and precision for a given material, set of measurement 

conditions, and Raman spectral model. 

The improved model of the CFM 

extends the transfer standard’s 

usefulness to a wide variety of gases. 

One of the domestic flow 

comparisons helped identify an 

important improvement for the 

company’s flow standard. 

Raman spectroscopic 

techniques provide a 

nondestructive means of 

determining the carrier 

concentration of 

semiconductor materials. 

Intensity (Arb. Units)

200 300 400 500 600 700

Raman Shift (cm-1)

 Measured Spectrum

 Fit Spectrum

L+

LLO

2O

CSTL scientists are examining the 

precision and accuracy of Raman 

spectroscopy to facilitate its use 

for semiconductor characterization. 
123

Preliminary Evaluation of Positive Secondary Ion Yield Enhancements Using 

Chlorodifluoromethane and Tetrachloroethene Backfilling 

P. Chi and J.G. Gillen (837) 

Positive secondary ion yields in Secondary Ion Mass Spectrometry (SIMS) are strongly enhanced 

by the presence of reactive gas species. Oxygen primary ion bombardment or oxygen gas 

backfilling of the analytical chamber is commonly used for this purpose. However, for some metal 

elements such as Nb, Mo, Ag, and Cd, oxygen bombardment or gas backfilling does not enhance 

the ion yields. Recently, experiments using SF5

+

 primary ions suggested that a fluorinated 

sample surface might provide some level of secondary ion signal enhancement. However, SF5

+

primary ion beams are not generally available on commercial SIMS instruments. This led us to 

explore alternative methods for 

enhancing ion yields by backfilling 

the sample surface with either 

chlorodifluoromethane (freon) or 

tetrachloroethene (C2Cl4) gas while 

depth profiling with an argon primary 

ion beam. 

The figure shows a depth profile of 

12 metal ion species at various freon 

backfill pressures. Most elemental 

secondary ion signals increased by 

one order of magnitude as the freon 

backfill pressure was increased from 

the 6.7x10-5 Pa to 2.7x10-4 Pa. 

Ion yield enhancements for 15 metal species under 2.7 x10-4

Pa freon and tetrachloroethene backfilling are listed in the 

table. The yields for Al, Fe, and Cu are lower while Si and Ni 

are unchanged under freon backfill as compared with 

oxygen backfill. However, the yield for Mg, Ti, V, Cr, Mn, Zn, 

Nb, Mo, Ag, and Cd with freon backfill was several times 

higher than that of oxygen. The yield enhancement for Mg, 

V, Cr, Mn, Zn, Nb, Mo, Ag, and Cd were also higher with 

tetradichloroethene backfill, but the yields for Al, Si, Ti, Fe, 

Ni, and Cu were slightly lower as compared with freon 

backfilling. The sputtering rate under freon and 

tetradichloroethene flooding is about 2.5 and 1.6 times 

faster, respectively, than oxygen flooding under similar 

sputtering conditions. 

Element Ion yields 

for Freon 

backfill 

norm. to 

O2 backfill 

Ion yields for 

Tetradichloroethene backfill 

norm. to O2 

backfill 

Mg 52 25.8 

Al 0.7 0.62 

Si 1 0.58 

Ti 2.5 0.97 

V 3.4 1.72 

Cr 2.8 1.31 

Fe 0.8 0.53 

Mn 2.7 1.40 

Ni 1.1 0.41 

Cu 0.4 0.35 

Zn 4.9 1.51 

Nb 8.9 1.50 

Mo N/A 3.22 

Ag 5.0 4.30 

Cd 10 7.70 

Use of either freon or tetradichloroethane gas 

backfilling was found to yield enhancements in 

secondary ion yields for many metallic elements, 

providing higher sensitivity for trace analysis by SIMS. 

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

0 400 800 1200 1600

Ti

V

Cr

M n

N i

C u

Z n

Z r

N b

M o

A g

C d

Tim e (s)

6.7x10-5 Pa

1.3x10-4 Pa

2.7x10-4 Pa

Freon Pressu re
124

Results of SiO2/Si Comparison between Neutron Scattering and grazing incidence x-ray 

photoelectron spectroscopy (GIXPS) 

T. Jach and E.W. Landree (837)

 

The metrology of gate dielectric films, an important 

consideration in complementary metal oxide 

semiconductor (CMOS) semiconductor fabrication, 

crosses a new measurement threshold when film 

thicknesses are less than 10 nm. The current 

thickness of these layers (and new materials being 

investigated to replace them) is of the order of 3 nm. 

Recent comparisons using established techniques, or 

even different instruments of the same type, indicate 

that there are uncertainties in the measurements of 

simple SiO2/Si films arising on this scale, which far 

exceed the supposed precision of the individual methods. In addition to the inherent need to 

reconcile disparate measurements of SiO2 films, the possibility for greater digressions arises as 

multilayer gate dielectrics are grown. Discrepancies between the results reported by different 

techniques begin to creep into measurements at the level of 

10 nm for three different reasons. Some may occur because 

different physical properties are actually measured and the 

“thickness” defined by these methods starts to differ as the 

scale decreases. Secondly, the assumed values of physical 

parameters determining a measurement may be inaccurate 

on a scale that was not previously of concern. Finally, some 

techniques employ processes that may change the sample 

on the scale of interest. We were able to test the first two 

possibilities with a comparison measurement between 

grazing incidence x-ray photoemission spectroscopy and 

neutron reflectivity. 

During the past year we were able to complete 

measurements on a set of samples of SiO2/Si in the 

thickness range of 3 nm to 7 nm. The methods 

employed, grazing incidence x-ray photoelectron 

spectroscopy and neutron reflectometry, are 

comparable techniques that rely on grazing angle 

beams scattering from thin surface layers. They 

differ, however, in relying on totally different physical 

interactions that depend, in turn, on different physical properties of the layer materials. Both 

methods provide values for the thicknesses of the layers, while being able to distinguish between 

the oxide layers and layers of contamination that may reside on the surface. We observe 

consistent differences between measurements made by the two methods. This is attributed partly 

to the difference in physical properties measured, and partly to the uncertainties about the correct 

physical values to use in determining thickness. 

The microelectronics industry is 

constrained to reduce the 

thickness of gate oxide layers as 

part of the overall scaling down 

of CMOS device dimensions. 

However, methods that would be 

used to measure the thicknesses 

on this scale are not in 

agreement.

Based on cited NIST results, GIXPS 

has been adopted as the method of 

choice for characterizing surface 

contamination on the Japanese 

research fabrication line dedicated to 

developing next-generation process 

technology (400 mm Si wafers). 
125

Thermophysical Properties of Gases used in Semiconductor Processing 

J.J. Hurly, K.A. Gillis, and M.R. Moldover (836) 

The semiconductor industry has identified process gases, 

“surrogate” gases, “carrier” gases, and their binary mixtures 

as the greatest area of need for accurate thermophysical 

property data. CSTL is measuring the thermophysical 

properties of gases widely used in semiconductor processing 

using novel, accurate, NIST-developed acoustic techniques. 

The properties include the speed of sound, ideal-gas 

heat-capacity, density (equation of state), viscosity, 

and thermal conductivity. Accuracy targets for 

thermophysical property data have been established 

that meet the needs for modeling chemical vapor 

deposition (CVD) processes and calibration of mass 

flow controller (MFC) using surrogate gases. 

In FY02, we completed speed of sound measurements 

in three surrogate gases and nine process gases. 

Typically, the standard relative uncertainty of the 

speed of sound was less than 0.01 %. The ideal-gas 

heat capacity was determined to within 0.1 % from the 

zero-pressure intercept of each isotherm. The slope 

and curvature of each isotherm provided information 

about each gas’s non-ideality from which we 

developed an equation of state to predict the gas’s 

density to within 0.1 %. The theory of the Greenspan 

acoustic viscometer was completed. Using this theory, the viscosity results for five test gases 

agreed within ±0.5 % with reference data. An acoustic resonator optimized to measure the 

thermal conductivity of surrogate gases and process gases has been designed and fabricated 

and is currently being characterized with gases of wellknown thermal conductivity. Measured data are 

disseminated as a database available via the internet. 

This database includes the heat capacity at constant 

pressure, thermal conductivity, viscosity, and a pressuredensity-temperature relation for process gases as well as 

the diffusion coefficients for the gaseous mixtures. 

The semiconductor industry 

has identified a significant 

need for accurate 

thermophysical property 

data for semiconductor 

processing gases. 

Improved thermophysical 

property data developed at 

NIST gives the semiconductor 

industry the capability to 

achieve process control with 

improved accuracy and 

product reproducibility. 

Thermophysical properties required 

to model generic mass flow controller 

(MFC) device components. 

http://properties.nist.gov/semiprop 
126
 127

12. Measurement Standards 

... supporting emerging technologies and the nation's measurement infrastructure 

NIST provides a wide variety of measurement, standards, and data services and programs to 

help U.S. industry improve its international competitiveness, commercialize new technology, and 

achieve total quality in all facets of business operations. CSTL maintains and disseminates 

measurement standards for chemical, biomolecular, and chemical engineering measurements by 

developing Standard Reference Materials (SRM®s), Standard Reference Data (SRD), and by 

providing Calibration Services. CSTL partners with NIST Technology Services to help 

disseminate the standards to NIST customers. 

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 2002 NIST sold approximately 31,000 

SRM units to about 6,600 unique customers. Approximately 17,000 of the units sold were from 

the ≈ 700 different types of materials that are certified for chemical composition. CSTL provides 

technical leadership for most of the chemical and compositional standards produced by NIST. 

SRMs are used for three main purposes: to 

help develop accurate methods of analysis 

(reference methods); to calibrate 

measurement systems; and to assure the 

long-term adequacy and integrity of 

measurement quality assurance programs. 

NIST SRMs also legally constitute part of 

the National Measurement System 

infrastructure of the United States and, as 

such, are essential transfer mechanisms for 

national as well as international 

measurement traceability. 

To meet the growing need for traceable standards, CSTL pioneered the NIST Traceable 

Reference Materials (NTRM) program. An NTRM is a commercially produced reference material 

with a well-defined traceability linkage to existing NIST 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 was 

implemented initially in the gas standards area to allow NIST to respond to increasing demands 

for high-quality reference materials needed to implement the “Emissions Trading” provisions of 

the Clean Air Act of 1990 (while facing the reality of constant human and financial resources at 

NIST). The program has been highly successful. Since its inception, 12 specialty gas companies 

have worked with NIST to certify over 9000 NTRM cylinders of gas mixtures that have been used 

to produce more than 500,000 NIST-traceable gas standards. A recent study conducted by RTI 

International estimates that the “net benefits” of the NTRM program projected 

through 2007 will be between $50M and $63M with a social rate of return of 

about 225%. The NTRM concept has been expanded to our Optical Filter 

Standards Program. Currently, this program is just getting underway with three vendors. 

Chemical Standards Development 

Examples of new standards in production in 

CSTL in several key areas: 

Health - Cardiac Troponin I 

Food - Nutraceuticals/Herbal Supplements

Environmental - Low Sulfur and Mercury Coal

Commodities - Suite of Cements, Sulfur in Diesel 

fuels, metals/metal alloys, Electronic Scrap 

Artifact Material (first SRM for recycling)

Forensics - Gunshot and Explosive Residues, 

and Chemical and Biological Weapons Detection.
 128

In addition to SRMs and SRD (discussed in a 

separate section), calibrations are utilized to 

disseminate measurement standards. CSTL 

plays a significant role in NIST’s Calibration 

Services, providing 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 FY 2002, there were 

1,687 test folders created, NIST-wide, for 

685 unique customers. CSTL served 174 

different companies and completed 

calibration services from 844 test folders. 

Instrumentation manufacturers and users 

rely on our measurement services to 

provide traceability to U.S. national 

measurement standards. 

Selected Chemical and Physical Standards Activities in FY 2002 

(Described in more detail in elsewhere in this report) 

Automotive and Aerospace

SRMs for Low Level Automotive Emissions 

W.J. Thorn III (839) 

SRMs for Hydrogen in Titanium 

R.M. Lindstrom, R.L. Paul, and H.H. Chen-Mayer (839) 

Chemical and Allied Products

Critical Benchmark Standards for the Cement Industry 

J.R. Sieber and A.F. Marlow (839); B.S. MacDonald and C.N. Fales (TS); S.D. Leigh and J.H. 

Yen (ITL); D. Broton (Construction Technology Laboratories); and S. A. Wilson (USGS)

Critical Benchmark Standards for the Metals Industry 

J.R. Sieber, A.F. Marlow, J.D. Fassett, G.C. Turk, R.M. Lindstrom, M.L. Salit, and M. 

Winchester (839); and J.R. Rumble (TS) 

Environmental Technologies 

SRMs for Contaminants on Air Particulate Matter 

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

Gravimetric Results of a Fine Particulate Carbonaceous Reference Material on QuartzFiber Filter 

G.A. Klouda (837) and J.J. Filliben (ITL) 

Physical Standards Development 

Examples of New Physical Standards under 

development in CSTL in the following core areas: 

Temperature - Primary Acoustic Thermometry

Pressure - Intrinsic Pressure Standards in the 

range 0.3 MPa to 5 Mpa 

Flow New Primary Gas Flow Standards decrease 

uncertainties more than a factor of 2 

Humidity - Rapid, low-cost methods for calibration 

of permeation tube generators 

Density - New Liquid Density Standards 
 129

Total Carbon Determination in Buffalo River Sediment Using Isotope Dilution Mass 

Spectrometry: Comparison of Techniques 

D.B. Klinedinst, R.M. Verkouteren (837); and K. Revesz (U.S. Geological Survey)

SRMs for Contaminants in Soils, Sediments and Tissues

C. Beck II, T.A. Butler, R.R. Greenberg, M. Howard, W.R. Kelly, J.R. Kucklick, R.M. 

Lindstrom, S.E. Long, E.A. Mackey, J.L. Mann, A.F. Marlow, K.E. Murphy, R. Oflaz, R.L. 

Paul, B.J. Porter, D.L. Poster, M.M. Schantz, J.R. Sieber, G.C. Turk, R.D. Vocke, S.A. Wise, 

L.J. Wood, L.L. Yu, R.L. Zeisler (839); and S. Tutschku (Universitat Leipzig, Germany) 

Standards to Support Stack Emissions Testing 

W.D. Dorko (839) 

Development and Critical Evaluation of a New ID/ICP-MS Method for Mercury in Sediments 

and Fossil Fuels 

S.E. Long and W.R. Kelly (839) 

Development and Critical Evaluation of New Methods for Determination of Methylmercury 

and Butyltin Species in Marine Tissues and Sediment 

M.M. Schantz, S.A. Wise (839); and S. Tutschku (Universitat Leipzig, Germany) 

Developing an Advanced Primary Ozone Photometer - A Collaboration with BIPM 

P.M. Chu, J.E. Norris, and F.R. Guenther (839) 

Food and Nutrition 

Completion of Suite of SRMs to Support Nutritional Labeling Issues 

K.E. Sharpless, J.M. Brown Thomas, S.A. Margolis, B.C. Nelson, C.S. Phinney, J.R. Sieber, 

and L.J. Wood (839) 

Reference Methods and Materials for the Chemical Characterization of Dietary 

Supplements 

S.A. Wise, K.W. Phinney, L.C. Sander, and K.E. Sharpless (839) 

Reference Methods and Standards for Biotech Foods

D.M. Bunk (839) 

Forensics and Homeland Security

Y Chromosome Assays and Standards Support Commercial Technologies 

J.M. Butler, R. Schoske, P.M. Vallone, J.W. Redman, and M.C. Kline (831) 

Standardization of Sample Preparation Techniques used in Analyzing Microorganisms by 

MALDI-TOF Mass Spectrometry 

C. Nelson, B.J. Porter, M.J. Welch, B.C. Nelson, E. White V (839); and W.G. Mallard (838) 

Verification of Calibration Procedure for Testing Military Gas Masks 

R.A. Fletcher, L.R. King, J.A. Small (837); M.R. Winchester (839); J.C. Yang and G.W. 

Mulholland (BFRL) 

New Measurement Technologies and Standards to Aid in Crime Scene Investigations 

W.A. MacCrehan (839); M. Layman, J. Secl, and A. Dorsey (George Washington Univ.) 
 130

Reference Methods and Standards for Alcohol and Drugs of Abuse Testing 

M.M. Schantz, J.V. Goodpaster, L.T. Sniegoski, S. Tai, and M.J. Welch (839) 

Health and Medical Technologies

The Complete Mitochondrial DNA (mtDNA) Genome Sequence of Human Cell Line HL-60 

and Its Inclusion in NIST’s mtDNA SRM 

B.C. Levin, D.K. Hancock, K.L. Richie (831); K.A. Holland (Gettysburg College); M. Coble 

and T. J. Parsons (Armed Forces DNA Identification Laboratory); L. J. Kienker (FBI 

Laboratory); and D.W. Williams (Georgia Bureau of Investigation)

Critical Evaluation of ID ICP-MS vs. TIMS as New National Reference Method for 

Electrolytes in Blood Serum 

K.E. Murphy, S.E. Long, J.D. Fassett, and M.S. Rearick (839) 

Newly Certified Trace Elements in Urine and Blood SRMs 

R.D. Vocke, C. Beck II, O. Ertas, W.R. Kelly, S.E. Long, and M.S. Rearick (839) 

Standards for in vitro Diagnostics and International Trade 

W.E. May, D.M. Bunk, J.D. Fassett, W.R. Kelly, S.E. Long, K.E. Murphy, B.C. Nelson, M.B. 

Satterfield, L.T. Sniegoski, S. Tai, R.D. Vocke, and M.J. Welch (839) 

Toward Comparable Cardiac Troponin I Measurements: Selection of a Primary Reference 

Material 

D.M. Bunk and M.J. Welch (839) 

Industrial and Analytical Instruments and Services

Atomic Standard of Pressure 

M.R. Moldover, J.W. Schmidt (836) and K. Szalewicz (University of Delaware) 

Development of High-Precision Isotope Reference Standards 

A.P. Miiller (836) 

Improved Vacuum Transfer Standards 

R.F. Chang (836)

International Standards for Calorimetry and Thermodynamics 

D.R. Kirklin and D.G. Archer (838) 

New SRMS for Near Infrared and Raman Spectroscopy 

W.S. Hurst, J.E. Maslar (836); E.S. Etz (837); and S.J. Choquette (839) 

Transducer-Assisted Piston Gauge Calibrations 

D.A. Olson (836) and T. Kobata (NMIJ, Japan) 

Microelectronics

Completion of Suite of Dopants in Silicon Standards 

R.L. Paul, R.R. Greenberg, R.M. Lindstrom (839); and D.S. Simons (837) 

Development of SiGe Compositional Standards for the Semiconductor Industry 

R.B. Marinenko, J.T. Armstrong, S. Turner, E. B. Steel, and D.S. Simons (837) 
 131

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 U.S. population 

have increased the need for demonstrating 

“traceability to NIST” and establishing a more formal 

means for documenting measurement comparability 

with standards laboratories of other nations and/or 

regions. 

NIST is the U.S. National Metrology 

Institute (NMI) and the agency of the 

U.S. Government responsible for U.S. 

efforts under the Treaty of the Metre. 

The Committee International des Poids 

et Mesures (CIPM), and its various 

consultative committees, organizes 

comparisons of national measurement 

standards. After review, the results of 

these comparisons are recorded in the 

BIPM database. In addition, coordination 

of similar efforts with Regional Metrology 

Organizations (RMOs) extend the 

comparison efforts to as many 

participants as practicable. 

In October 1999, NIST 

signed the CIPM Mutual 

Recognition Arrangement (MRA). 

Since 1999 CSTL has systematically compared U.S. national measurement standards to 

establish degrees of equivalence of U.S. national measurement standard with those of other 

NMIs. These international activities add value to NIST standards and services, particularly for our 

customers involved in international trade. These MRA-related activities guarantee recognition of 

U.S. standards by U.S. trading partners. 

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/ 
 132

Systema Interamericano de Metrologia (SIM) is the RMO that includes the United 

States. The metrology organizations that make up SIM are known as: NORAMET 

(North America), CAMET (Central America), CARIMET (Carribean), ANDIMET 

(Andes Region), and SURAMET (South America). Regional cooperation leads to a 

wider harmonization of measurements and standards, facilitates free flow of trade, 

and is a necessary first step to globalization. NIST and National Research Council 

(NRC) Canada link SIM countries to the BIPM, 

and as such, provide greater opportunities for 

international trade and commerce. CSTL staff 

provides leadership for SIM by chairing the 

Chemical Metrology Working Group of SIM, and 

by serving as the U.S. respresentative to the 

JCRB, in order to assure the effective, fair, and 

metrologically sound implementation of the MRA. 

CIPM Consultative Committees

The CIPM has set up a number of Consultative Committees, which bring together the world's 

experts in their specified fields as advisers on scientific and technical matters. Among the tasks of 

these Committees are the detailed consideration of advances in physics that directly influence 

metrology, the preparation of Recommendations for discussion at the CIPM, the identification, 

planning and execution of key comparisons of national measurement standards, and the 

provision of advice to the CIPM on the scientific work in the laboratories of the BIPM. 

CIPM Consultative Committees for Temperature (CCT) and Mass (CCM) 

Researchers in CSTL’s Process Measurements Division have significant participation in the CC’s 

for Temperature (CCT) and Mass (CCM). CSTL has lead or participated in many Key 

Comparisons (KCs) in the past several years. In thermometry, these efforts have resulted in 

establishing equivalence levels over the ITS-90 range from 14 K to 1235 K. KCs organized by the 

CCM include Division activities in both the pressure and vacuum and the flow project areas. In 

the pressure and vacuum project the Division leads or participates in KCs that cover the pressure 

range 3x10-6 Pa to 500 MPa. NIST has piloted three CCM Key Comparisons in the last several 

years, completing two in FY02 that demonstrated general equivalence among the participants, 

revealed no systematic bias between alternative realizations of the Pascal, were the only CCM 

KCs completed on schedule, and set the standard for the manner in which KCs should be 

conducted. In addition, CSTL researcher G. E. Mattingly chairs the CCM’s Working Group for 

Fluid Flow (WGFF). The WGFF has formed its strategy, structure, and schedule for comparisons 

in six areas: water; hydrocarbon liquids; low-pressure air and high-pressure; high-flow natural 

gas; air speed; and liquid volume. The WGFF chair, working closely with the NIST Statistical 

Engineering Division, has developed a sound, statistically based experimental design and 

supporting analysis method applicable to all KCs. The design of these protocols and transfer 

standards will set precedents that should advance the state-of-the-art in conducting all future flow 

laboratory comparisons. This metrological plan was presented to the CCM in May 2002. The 

BIPM Director noted that this plan should result in a unique comparability of the flow standards in 

the participating NMIs and fulfill “the ideal of the MRA”. NIST will pilot the KC for the lowpressure gas flow. Recent advances in U.S. standards and procedures (described below) are 

expected to materially improve the performance-level of this KC. 

CSTL’s Analytical Chemistry Division has 

led the critical review of CMC data for 

Appendix C for the BIPM Database. SIM has 

approximately 1140 CMC entries scheduled 

for inclusion in the BIPM database. By the 

beginning of 2003, the BIPM database will 

include approximately 3000 CMCs for 

chemistry. About 1000 will be from NIST.
 133

International Comparisons of Pressure Standards (CCM Key Comparisons) 

A.P. Miiller, P.J. Abbott, and A. Lee (836) 

The status of NIST participation in eight international comparisons of pressure and vacuum 

standards, at the highest metrological level, is presented. These comparisons help establish 

degrees of equivalence of national standards from 

3x10-6 Pa to 500 MPa. NIST has piloted three CCM 

Key Comparisons (two to completion in FY02). The 

completed comparisons were the first successful 

international comparisons in this pressure range (1 Pa 

to 1000 Pa, absolute and differential modes), and 

demonstrated general equivalence among the 

participants, revealed no systematic bias between 

alternative realizations of the Pascal, and were the only CCM pressure comparisons completed 

on time. The measurement phase of the third comparison (3x10-6 Pa to 9x10-4 Pa) has been 

completed and is in the analysis and reporting phase. 

Comparisons CCM.P-K4 and CCM.P-K5 

were formally approved by the CCM in 

FY02. In recognition for overcoming major 

technical and engineering barriers in 

developing robust, state-of-the-art low-pressure transfer standards that enabled the successful 

completion of these two precedent-setting comparisons, A. P. Miiller was awarded the 2002 

CSTL Technical Achievement Award. He was also appointed to the Chair of the Low-Pressure 

Working Group of the CCM. Comparison CCM.P-K3 is also piloted by NIST. The transferstandard package that was developed at NIST consists of two spinning rotor gauges, three 

Bayard-Alpert ionization gauges, and began circulation in FY99. The comparison is behind due 

to delays between participants, but measurements were completed in FY02, although some 

participants have not yet submitted their results. 

 

To help alleviate technical trade 

barriers, the relative agreement of 

national pressure and vacuum 

measurement standards needs to 

be assessed, established, formally 

recognized, and maintained. 

CCM.P-K4 and -K5 have formally 

established the degree of equivalence 

between several NMIs in an industrially 

important pressure range. Despite over a 

decade of attempts, -K4 and -K5 are the 

first successful comparisons in this range, 

have unambiguously established degrees 

of equivalence between NMIs, and revealed 

no significant relative bias among the 

different techniques to realize the Pascal 

from 1 Pa to 1000 Pa. The innovative 

transfer package developed for this 

comparison has formed the basis for many

variants in regional comparisons 

throughout the world. 

The purpose of intercomparisons is to establish degrees of equivalence between 

national pressure and vacuum standards for pressures from 3x10-6 Pa to 500 MPa. 

International trade is, in part, based upon the equivalence of measurements. Some 

of the largest segments of industrial measurements are in the areas of pressure 

and vacuum. 

Results for CCM.P-K4: shows the 

deviation of measurements at NMIs from 

the reference value divided by the 

uncertainty (95% confidence level). 

-2

-1

0

1

2

1 10 100 1000

Pressure / Pa

(D j = p j - p R ) / U j

IMGC

MSL-NZ

NIST

NPL-UK

Dj = Uj

Dj = - Uj
 134

A Summary of CCM Key Comparisons in Pressure 

Comparison 

Number 

Pressure 

Range 

Transfer 

Standard Status 

Pilot 

NMI* Participants* 

CCM.P-K1.a, 

b 

0.05 - 1 MPa 

(gauge) 

Piston 

Gauge 

Approved a-6 

b-3 

1ab, 2a, 3ab, 

5ab, 6ab 

CCM.P-K1.c 0.08 - 7 MPa 

(gauge) 

Piston 

Gauge 

Approved 5 1, 2, 3, 4, 5, 6 

CCM.P-K2 10 – 120 kPa 

(absolute) 

Piston 

Gauge 

Analysis & 

Reporting 

2 1, 2, 5, 6, 10, 12, 

13, 14, 15, 16, 

17, 18 

CCM.P-K3 3x10-6 - 9x10-4 Pa 

(absolute) 

Spinning 

Rotor and 

Ion Gauges 

Analysis & 

Reporting 

1 1, 2, 5, 7, 8, 9 

CCM.P-K4 1 - 1000 Pa 

(absolute) 

Low 

Pressure 

Transducers 

Approved 1 1, 2, 5, 7, 8, 9, 

10 

CCM.P-K5 1 – 1000 Pa 

(differential) 

Low 

Pressure 

Transducers 

Approved 1 1, 2, 5, 10, 11 

CCM.P-K6 10 – 120 kPa 

(differential) 

Piston 

Gauge 

Analysis & 

Reporting 

2 1, 2, 5, 6, 10, 13, 

14, 15, 16, 17, 

18 

* 

List of NMIs: 

1. National Institute of Standards and Technology (NIST) – U.S 

2. National Physical Laboratory (NPL) – United Kingdom 

3. Laboratoire National D’Essais (LNE) – France 

4. National Research Laboratory for Metrology (NRLM) – Japan 

5. Instituto di Metrologia “G Colonnetti” (IMGC) – Italy 

6. Physikalisch-Technische Bundesanstalt (PTB) – Braunschweig, Germany 

7. Physikalisch-Technische Bundesanstalt (PTB) – Berlin, Germany 

8. Korean Institute of Standards and Science (KRISS) – S. Korea 

9. National Physical Laboratory (NPLI) – India 

10. Commonwealth Scientific and Industrial Research Organization (CSIRO-NML) – Australia 

11. Measurement Standards Laboratory (MSL) – New Zealand 

12. Bureau International des Poids et Mesures (BIPM) 

13. Insitut National de Metrologie (INM) – France 

14. National Research Council (NRC) – Canada 

15. Nederlands Meetinstituut (NMi) – Netherlands 

16. Office Fédéral de Métrologie (OFMET) – Switzerland 

17. National Institute of Metrology (NIM) – China 

18. D.I. Mendeleyev Institute for Metrology (VNIIM) – Russian Federation 
 135

Consultative Committee for Amount of Substance (CCQM) 

NIST has taken a leadership role in the International Committee of Weights and MeasuresConsultative Committee on the Quantity of Material (CCQM) and the Chemical Metrology 

Working Group of the Interamerican System for Metrology (SIM) in order to assure the effective, 

fair and metrologically sound implementation of this MRA. The CCQM has formed seven working 

groups: (1) gas analysis, (2) organic analysis, (3) inorganic analysis, (4) electrochemistry, (5) 

biometrology, (6) surface analysis and (7) key comparisons. 

These working groups are responsible for selecting and 

overseeing the operation of key comparisons that address 

chemical measurement-related issues important for international 

trade, environmental, health, and safety-related decision 

making. CSTL’s Analytical Chemistry Division is leading various 

activities within five of the seven working groups and chairing 

the Organic Analysis Working Group. During the past five years, 

CSTL has participated in 46 of the 53 comparison studies 

conducted under the auspices of the CCQM, and served as Pilot 

Laboratory in 18. An additional 36 studies are planned for the 

next three years, and NIST has committed to pilot 9 of these. A 

full listing and status report for recent KCs are provided below. 

CSTL’s ACD is also 

working with CITAC 

(Cooperation in 

International Traceability 

in Analytical Chemistry) to 

establish practical, yet 

metrologically sound, 

vertical traceability links 

between the NMIs and 

chemical testing labs in 

various countries and 

regions around the world. 

CCQM Key Comparisons

• CCQM K-1, CO, CO2, NO, and SO2 in N 2, and natural gas

• CCQM K-2, Cd, Pb in natural water

• CCQM K-3, CO, CO2, propane in N 2

• CCQM K-4, Ethanol in air

• CCQM K-5, DDE in Fish Oil

• CCQM K-6, Cholesterol in Serum

• CCQM K-7, BTX in N 2/air

• CCQM K-8, Elemental solutions standards

• CCQM K-9, pH 7 (phosphate)

• CCQM K-10, BTX in air (ng/g level)

• CCQM K-13, Pb, Cd in sediments

• CCQM K-29, Zn in Rice

• CCQM K-11, Glucose in Serum, Draft B report

• CCQM K-12, Creatinine in Serum, Draft B report

• CCQM K-16, Natural gas, Draft A report

• CCQM K-17, pH 4.1 (phthalate), Draft A report

• CCQM K-21, DDT in Fish Oil, Draft B report

• CCQM K-24, Cd in Rice, Draft B report

• CCQM K-25, PCBs in Sediments, Draft A report

Completed

Reports 

Drafted
 136

Selected CCQM KCs Completed in FY 2002 

International Benchmarking of NIST’s Classical and Electroanalytical Measurement 

Capabilities 

K.W. Pratt (839) 

In FY 2002, NIST participated in 4 CCQM pilot studies connected with international benchmarking 

of classical (assay) techniques, pH metrology, and anion solutions. Each study was designed to 

assess the factors that govern the agreement attained among the participants [national metrology 

institutes (NMIs)] for the given measurand. The 

results of each study indicated that the NIST 

measurements are at the state of the art for the 

respective measurement. Results for the pH and 

assay studies also revealed sources of systematic 

bias that are difficult to detect using results 

obtained at a single NMI. 

Each pilot study evaluated the degree of agreement 

obtained for the given measurand for each NMI. 

The pilot study in most cases served as a 

preliminary trial to a future key comparison (KC), 

the quantitative results of which will fix the 

international acceptance of measurements by each 

NMI (including NIST). Also, the pilot studies 

investigated systematic, uncontrolled sources of 

bias that limit the agreement obtained between 

NMIs. This second justification utilizes the 

information obtained from different NMIs 

performing a given primary measurement 

using their own realization to obtain 

information not attainable by a single NMI 

performing its primary measurement alone. 

The pH pilot study (CCQM-P37) was 

performed in an effort to elucidate 

systematic effects that contributed to 

unexpectedly large deviations of certain 

NMIs (not including NIST) in the previous 

pH KC, CCQM-K17. 

The HCl assay pilot study (CCQM-P19.1) 

was undertaken to minimize the effects of packaging and shipping that were observed in the HCl 

solution distributed to the participants in CCQM-P19, the previous study. CCQM-P19.1 also had a 

wider range of NMIs participating and therefore included a better evaluation of H+

 and Cl- assay 

methods. NIST was the coordinating laboratory. 

CCQM-P36 [potassium acid phthalate (KHP) assay] provided information on the importance of 

CO2 interference in assay of this widely used acidimetric standard and exposed a bias of 

approximately +0.04 % at NIST that had arisen since the latest KHP SRM certification. 

CCQM-P19.1 (HCl assay) vindicated the use of improved packaging to eliminate the influence of 

packaging on participants' results and provided the first statistically useful comparison of H+

 and 

Cl-

 assays. CCQM-P37 (fundamental pH study) revealed obscure factors connected with primary 

pH (Harned) cell design and usage that should result in improved uncertainties for future NIST pH 

Nominal 0.1 S/m (1000 μS/cm)

CCQM-P22: Conductivity P22: Conductivity 

(primary and secondary measurements) (primary and secondary measurements)

Study Period: 2001 Study Period: 2001

1 2 3 4 6 7 8 9 10 11 12 13

NMI

CCQM-K17: pH of Phthalate Buffer Solutions K17: pH of Phthalate Buffer Solutions

pH nominal value: 6.9 at 25 °C Dates of Study: 2001 pH nominal value: 6.9 at 25 °C Dates of Study: 2001 

[15 °C, 25 °C, 37 °C] [15 °C, 25 °C, 37 °C] 

pH @ 25°C

3.995

4

4.005

4.01

4.015

4.02

4.025

CENAM

CMI

KRISS

AIST

NRCCRM

DPLPTB

NIST

SMU

GUM

VNIIFTRI

pH

Data in Draft Form

1 2 3 4 5 6 7 NIST 9 10 11

NMI

CCQM-K17: pH of Phthalate Buffer Solutions K17: pH of Phthalate Buffer Solutions

pH nominal value: 6.9 at 25 °C Dates of Study: 2001 pH nominal value: 6.9 at 25 °C Dates of Study: 2001 

[15 °C, 25 °C, 37 °C] [15 °C, 25 °C, 37 °C] 

pH @ 25°C

3.995

4

4.005

4.01

4.015

4.02

4.025

CENAM

CMI

KRISS

AIST

NRCCRM

DPLPTB

NIST

SMU

GUM

VNIIFTRI

pH

Data in Draft Form

1 2 3 4 5 6 7 NIST 9 10 11

NMI

pH @ 25°C

3.995

4

4.005

4.01

4.015

4.02

4.025

CENAM

CMI

KRISS

AIST

NRCCRM

DPLPTB

NIST

SMU

GUM

VNIIFTRI

pH

Data in Draft Form

1 2 3 4 5 6 7 NIST 9 10 11

NMI
 137

SRMs. The CCQM-P32 results (PO4

3- and Cl-

 anion solutions) supported the NIST claims for 

measurement capability for the respective measurements. 

The results of the pilot studies in KHP assay and anion solutions provided the first estimate under 

CCQM auspices of the international agreement that is attainable for the respective measurands. 

These pilot studies are likely to result in corresponding future KCs that underpin the claims of 

measurement capability for the participating NMIs (including NIST). 

The results of the HCl assay and fundamental pH studies provided essential information 

regarding primary pH measurements and systematic sources of uncertainty that impact all 

primary pH measurements. These results directly impact details of the methodology used by the 

NMIs participating in already-planned future pH KCs. 

International Benchmarking of CSTL Capabilities in Gas Metrology 

F.R. Guenther (839) 

Over the past year, NIST participated in CCQM-P23 Preparation of Gravimetric Gas Standards 

Pilot Study and CCQM-K16 Natural Gas Key Comparison. As the year ends we are also 

participating in CCQM-P41 Greenhouse Gases Pilot Study, and working with NMIJ (Japan) on 

the prototype for the CCQM-K22 volatile organic chemicals (VOCs) Key Comparison. Each study 

was designed to assess the comparability attained among the participants [national metrology 

institutes (NMIs)] for the given measurand. The results of each study indicated that the NIST 

measurements are at the state of the art for the respective measurement. Results for CCQM-P23 

revealed a bias among the NMIs which was traced back to the isotopic abundance of 13C in the 

pure carbon monoxide that used to prepare the gas standards. Once this difference was factored 

into the results, all the NMIs were in good agreement. 

Each study evaluated the degree 

of agreement obtained for the 

given measurand for each NMI. 

The pilot studies in most cases 

serve as a preliminary trial to a 

future key comparison (KC), the 

quantitative results of which will fix 

the international acceptance of 

measurements by each NMI 

(including NIST). Also, the pilot 

studies investigated systematic, 

uncontrolled sources of bias that 

limit the agreement obtained 

between NMIs. This second 

justification utilizes the information 

obtained from different NMIs 

performing a given primary 

measurement using their own realization to obtain information not attainable by a single NMI 

performing its primary measurement alone 

The CCQM-P23 Preparation of Gravimetric Gas Standard Pilot Study was intended to study the 

source of uncertainty in the preparation of primary gas standards. Carbon monoxide was chosen 

because it was thought that this measurand was not difficult to handle and analyze. Thus it 

should be relatively straightforward to determine small uncertainty sources. The results showed 

major biases between the NMIs, especially NIST and KRISS (Korea). After much work at NIST 

and several other NMIs, the source of the bias was traced to differences in the 13C abundance of 

CCQM-K16a: Gas mixture K16a: Gas mixture - Natural Gas Type IV (low calorific mixture) Natural Gas Type IV (low calorific mixture) 

Period of Study: 2001 12 measurands

NIST is Lab “H”

Nominal composition: Nitrogen 12.0 %, mol/mol Nominal composition: Carbon dioxide 4.0 %, mol/mol
 138

the pure carbon monoxide (CO). It turns out that gas suppliers strip out the 13C CO from the pure 

gas to sell to producers of isotopically pure organic compounds. The result is that primary 

standards produced with the depleted 13C CO will show a bias on certain non-dispersive infrared 

analyzers (NDIR). The bias is not seen in gas chromatographic methods. This result has major 

implications in the automotive industry, where measurements are needed to within 1 % of the true 

value to meet regulations. 

The CCQM-K16 Natural Gas Key Comparison 

was intended to measure the comparability of 

Natural gas measurements at the participating 

NMIs. Unlike past comparisons, this natural 

gas included the heavy hydrocarbons. Due to 

shipping problems, NIST was able to analyze 

only one of the two cylinders sent to 

participants. NIST’s results show excellent 

agreement with the majority of the NMIs. A 

similar Pilot Study was organized by NIST for 

the SIM countries. This study indicated that 

there remains much work to be done in SIM to 

bring the comparability among SIM NMIs in line 

with the CCQM. NIST is working with NMIJ in 

Japan on the proposed CCQM-K22 Volatile Organic Compounds (VOC) in Air. 

International Benchmarking of CSTL Inorganic Analytical Measurement Capabilities 

R.R. Greenberg, J.D. Fassett, K.S. Murphy, R. Zeisler, E.A. Mackey, L.L. Yu, L.J. Wood, J.R. 

Sieber, and A.F. Marlow (839) 

NIST/CSTL successfully participated in five 

pilot and key comparisons of the inorganic 

working group of the CCQM this year: 

CCQM-P11 Arsenic in Shellfish, CCQMK24 Cadmium in Rice, CCQM-P29 Zinc in 

Rice, CCQM-P13 Synthetic Food Digest, 

and CCQM-P25 Minor Elements in Steel. 

NIST led the pilot study on Arsenic in 

Shellfish, which was based on an oyster 

tissue similar to SRM 1566b. Arsenic is a 

toxic element and, thus, its determination in 

oyster tissue, a food matrix, is of general concern. Results were received from eleven 

laboratories from nine countries (see figure). 

The NIST results of (0.1287 ± 0.0015) μmol/g 

were in excellent agreement with the mean of 

(0.1279 ± 0.0016) μmol/g determined from all 

participants except one, who apparently had 

problems with their analysis. After the results 

became available, the CCQM decided to 

continue on to a key comparison for Arsenic 

in Shellfish (CCQM-K31). 

The rice comparison was split into both a key 

and pilot comparison. Thus, for those 

participants who felt comfortable with their Cd 

measurement capabilities, they could register 

for the key comparison CCQM-K24. 

result 11

NIST

CCQM-P11: Arsenic in Shellfish

Study Period: 2001 Study Period: 2001

1 2 3 4 5 6 7 8 9 10 11

result 11

NIST

CCQM-P11: Arsenic in Shellfish

Study Period: 2001 Study Period: 2001

1 2 3 4 5 6 7 8 9 10 11

Presently the CCQM-P41 Greenhouse Gases 

Pilot Study is proceeding, and will be 

completed by early 2003. This study will 

demonstrate the comparability of the NMIs 

for methane and carbon dioxide. This will 

set the stage for future comparisons in 

global warming gases, and should enable 

efforts to mitigate and control emission of 

these gases internationally. The CCQM is 

working closely with the World 

Meteorological Organization to link the 

measurement of these gases in the 

atmosphere to the SI. 

Key comparisons are an important method 

of demonstrating the claimed measurement 

capabilities of national metrology institutes 

(NMIs), as well as establishing the 

equivalence of measurements made by 

different NMIs. To serve US interests, it is 

important that NIST successfully participates 

in relevant measurement comparisons under 

the CIPM Mutual Recognition Arrangement. 
 139

The same material was used for a 

pilot comparison CCQM-P29 Zn 

and Cd in rice. We chose to 

participate in the key comparison; 

the NIST ID-ICP-MS and INAA 

determinations were combined 

and reported. This result was in 

excellent agreement with the 10 

other laboratories [NIST: (14.36 ± 

0.23) nmol/g; CCQM: (14.40 ± 

0.09) nmol/g (see figure). For the 

pilot comparison, the two NIST Zn 

results (ID-ICP-MS and INAA) 

were reported separately. 

Likewise, the Zn results were in excellent agreement [NIST ID-ICP-MS: (0.3554 ± 0.0067) μmol/g; 

NIST INAA: (0.3559 ± 0.0095) μmol/g; CCQM: (0.3527 ± 0.0024) μmol/g]. The major source of 

uncertainty for all NIST measurements in both K24 and P29 was in achieving a dry-mass basis 

weight. It was recognized that the CCQM-P29 Zn data was of “key comparison” quality as well. 

Rather than continue on to a key comparison, it was decided to accept that “the light of K24 

shines at least as far as Zn.” Cadmium in rice represents a toxic element in food; zinc is a 

nutritional element. 

The Pilot Study CCQM-P13 Metals in Artificial Food Digest was proposed by LGC to benchmark 

measurement of nutritional, essential, and toxic elements in foods. They proposed to avoid the 

difficulty in assigning a consensus key comparison reference value by spiking a synthetic matrix 

solution with gravimetrically known amounts of the target analytes: Ca (nutritional), Cu (essential) 

and Cd (toxic). Thus, issues associated with both dissolution and homogeneity were avoided. 

We decided to determine only the elements Ca and Cu; we felt that our capabilities for Cd had 

been demonstrated adequately in previous studies. We have been applying ICP-OES 

methodology with standard additions calibration in our recent certification measurements of food. 

(This year we made certification measurements for SRM 2387 Peanut Butter, SRM 2385 

Spinach, and Milk Powder.) Thus, we felt it was most appropriate that this method be used in the 

Pilot Study. The results for Ca and Cu were +1.3 % and –0.8 % relative to the reference values, 

respectively. The average deviations from the reference values were –0.5 % and +3.5 % 

respectively. The observation was made that the laboratories using IDMS had larger deviations, 

-2.1 % and +3.8 %, illustrating again that it is not the technique that endows accuracy but the skill 

of the laboratories that apply the technique. 

The first metal and metal alloy study evolved from proposals by 

NMIJ (Japan), BAM (Germany), and NIST. Designated CCQMP25, it is for minor elements (Cr, Mo, Ni, and Mn) in a low-alloy 

steel. These elements control specific steel properties. ACD 

determined all elements in the steel using its recently developed 

matrix independent XRF borate fusion method, and three 

elements (Cr, Mn, and Mo) were determined by INAA. We also 

coordinated the submission of results from 4 U.S. industry 

laboratories. The end-of-year deadline for this study meant that 

comparative results have not yet been formally revealed. 

However, results were shown graphically at the Fall 2002 

CCQM Inorganic Working Group meeting, and the NIST results 

were consistent with those submitted by other NMIs. 

CSIR

NRC

NRCCRM

IRMM

BAM

NMi NIST NARL

NMIJ EMPA

LGC

KRISS

13.2

13.4

13.6

13.8

14.0

14.2

14.4

14.6

14.8

15.0

15.2

 c 

nmol·g-1

CCQM-K24: Cd in rice

KCRV: 14.418 ± 0.087 nmol·g-1 ;U=ku c , k =2

The metal and metal alloy 

industries are huge and 

the economic impact of 

chemical measurements is 

very large. The range of 

commercial metal and 

alloy materials is also very 

large and, thus, it has 

been a challenge to 

identify the appropriate 

measurement activity for 

CCQM in this area. 
 140

Strategic Bilateral Collaborations and Intercomparisons with NMIs 

W.E. May, F.R. Guenther, S.A. Wise, and R.R. Greenberg (839) 

In addition to these global and regional activities described in this report, we are also establishing 

a limited number of strategic bilateral collaborations and intercomparisons with NMIs around the 

world. For example, our collaboration with the Netherlands Measurement Institute (NMi) for 

determining the equivalence of primary gas standards has resulted in a formal 

“Declaration of Equivalence” that is recognized by the U.S. EPA and European 

environmental regulatory bodies as documenting the equivalence between seven 

NIST and NMi primary gas mixture suites. A bilateral comparison of optical 

transmittance at five visible wavelengths and for transmittance levels between 1.0 

and 10-3 is currently underway between NIST and the National Physical Laboratory 

(NPL) of the UK. This is the first international comparison for the second-generation CSTL 

reference spectrophotometer and is a precursor to an anticipated 

Memorandum of Cooperation and Declaration of Equivalence between 

NIST/CSTL and NPL with respect to 

optical transmittance. Equivalence 

between the NIST and NPL 

transmittance scales is expected to 

ease the regulatory traceability 

burden for end users, especially 

multinational pharmaceutical companies. Our formal 

agreement with NRC-Canada (via the NAFTA Treaty) for 

cooperation in marine environmental studies has fostered 

collaborations in the certification of a several certified 

reference materials important for trade and environmental 

decision-making between the U.S. and Canada. We have a Cooperative Arrangement with NIMC 

(Japan) for collaborative efforts in the area of pure volatile organic compound standards. These 

highly pure reference compounds will serve as 

primary references for many of our gas mixture 

SRMs and our quantitative reference database 

(SRD-79). Several additional strategic bilateral 

arrangements with other National Metrology 

Institutes and standards laboratories are being 

discussed. 

CSTL continues to work with 

CITAC (Cooperation in 

International Traceability in 

Analytical Chemistry) to 

establish practical, yet 

metrologically sound, vertical 

traceability links between the 

NMIs and chemical testing labs 

in the various countries and 

regions around the world. 
141

13. Data and Informatics 

... assuring that U.S. Industry has access to accurate and reliable data, 

 and predictive models 

One of CSTL’s goals is to assure that U.S. industry has access to 

accurate and reliable data and predictive models to determine the 

chemical and physical properties of materials and processes. CSTL’s data and informatics 

activities impact all industry sectors from biotechnology and microelectronics to energy and 

instrument manufacturers. The NIST Standard Reference Data (SRDs) series has grown to 

over 50 electronic databases in chemistry, physics, materials, building and fire research, software 

recognition, and electronics. Versatile interactive databases provide easy access to high quality 

NIST data. Many databases are now available via the World Wide Web. 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 in the sections below, and a full listing 

of activities, with references to the appropriate program section, is also provided. 

During 2002, a new version of the NIST Chemistry WebBook was released. The WebBook 

remains one of the most widely used resources for chemical and physical property data, and 

usage is expected to grow at about 20% per year. During the past year, over 500,000 distinct IP 

addresses accessed the available information. The wide variety of users in industry, academia, 

and government, and the high rate of return users attest to the value of this resource. The 

WebBook will also be linked to NIST SRDs such as the NDRL/NIST Solution Kinetics Database

that are now accessible on the Internet. 

Another significant achievement in 2002 was the completion of a new version of the NIST Mass 

Spectral Library. The library now includes over one-third more spectra, and some of the spectra 

formerly in the library were replaced with higher-quality replicates or newly acquired spectra. 

These improvements will enhance the reliability of the library for identifying unknown, volatile 

chemical compounds by GC-MS. Approximately 2,500 new libraries are installed on instruments 

each year. 

An updated version of the Reference Fluid Thermodynamic and Transport Properties 

(REFPROP) database was also recently released. This database provides industrial users with 

the most accurate properties available for pure fluids and fluid mixtures used in refrigeration and 

air conditioning systems. The new version includes additional fluids and mixtures, improved 

property models, increased calculational speed, and an enhanced user interface. 

The Protein Data Bank (PDB) is a unique worldwide repository for three-dimensional structural 

data for biological macromolecules. The program was initiated in 1971 at Brookhaven National 

Laboratory and is now managed by the Research Collaboratory for Structural Bioinformatics 

(RCSB), whose three members include: Rutgers, The State University of New Jersey; the San 

Diego Supercomputer Center at the University of California; and the National Institute of 

Standards and Technology. Currently, there are nearly 20,000 entries in the archive, and many 

secondary sources of information are derived from PDB data. Uniformity of structural data is 

crucial for meaningful application of these data in the pharmaceutical and biotechnology 

industries, and NIST has played an important role in developing data standards that ensure 

global uniformity and facilitate data exchange. 
142

Summary of Data and Informatics Activities for FY 2002 

(Described in more detail in elsewhere in this report)

Pharmaceuticals and Biomanufacturing 

The Protein Data Bank 

T.N. Bhat, G.L Gilliland (831); and P. Fagan (TS) 

Target Site of Intron Gain Inferred by a System for Phyloinformatic Analysis (SPAN) 

A.B. Stoltzfus (831/CARB), and W-G. Qiu (CARB/UMBI); and N. Schisler (Pomona College), 

Theoretical Studies of Enzyme Mechanisms 

M. Krauss (831) 

Chemical and Allied Products 

Notre Dame Radiation Laboratory (NDRL)/NIST Solution Kinetics Database on the Web 

T.C. Allison, R.E. Huie, and P. Neta (838)

Photochemical Reduction of CO2 Catalyzed by Metalloporphyrins 

P. Neta (838) and J. Grodkowski (Institute of Nuclear Chemistry and Technology, Warsaw) 

An Efficient Model to Compute the Exact Exchange Potential in Density Functional Theory 

C.A. Gonzalez and T.C. Allison (838); E. Ludena, V. Karasiev, and I. Gonzalez (Instituto 

Venezolano de Investigaciones CientÌficas); and Y. SimÛn-Manso (Universidad de Chile) 

Environmental Control of α-Cr2O3 (0001) Surface Chemistry 

A.M. Chaka (838), N. Harrison (Imperial College and Daresbury Laboratory), 

A. Wander (Daresbury Laboratory), and X.-G. Wang (GM Research Laboratories) 

Cylinder Filling Ratios for Compressed Liquids 

M.L. Huber and D.G. Friend (838) 

A Standard Chemical Identifier 

D. Tchekhovskoi and S.E. Stein (838) 

The NIST WebBook - NIST Chemical Reference Data for Industry 

P.J. Linstrom and W.G. Mallard (838) 

Thermodynamics Research Center (TRC) Comprehensive Program on Critical Data 

Evaluation

M.L. Frenkel, R.D. Chirico, Q. Dong, G.R. Hardin, and R.A. Stevenson (838); 

X. Yan and X. Hong (Contractors); and V.V. Diky (Belarusian State U., Belarus) 

Measurements, Modeling and Data for Pressure-Driven Membrane Separations 

C.D. Muzny, K. Benko, and J.L. Scott (838); M. Chapman-Wilbert and K. Price (U.S. Bureau 

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

Gas Chromatography Retention Index Library: A Major New Resource Nears Completion 

J.K. Klassen, P.J. Linstrom, and S.E. Stein (838) 

The NIST Mass Spectral Library: A Major Update 

A. Mikaya, S.E. Stein, D.V. Tchekhovskoi, C.L. Clifton, and Q.-L. Pu (838) 
143

Industrial Fluid Properties Simulation Challenge: Benchmark Standards 

D.G. Friend, J.W. Magee, A.M. Chaka, R.D. Mountain, R.D. Johnson III, L.A. Watts, E.W. 

Lemmon, R.D. Chirico, M.O. McLinden, A. Laesecke, and T.J. Bruno (838) 

Properties and Processes for Cryogenic Refrigeration 

R. Radebaugh, P.E. Bradley, and M.A. Lewis (838); R. Gates (Univ. of Colorado); L. 

Reynolds (Univ. of Colorado; J. Gary and A. O'Gallagher (ITL); and J. Pfotenhauer (Univ. of 

Wisconsin) 

Energy Systems 

Properties of Refrigerant Mixtures Near the Critical Point 

M.O. McLinden, R.A. Perkins, E.W. Lemmon, A. Laesecke, and J.W. Magee (838) 

Release of Version 7.0 of the NIST REFPROP Database 

E.W. Lemmon, M.O. McLinden, and M.L. Huber (838)

Viscosity Measurements and Model Comparisons for Second-Generation Alternative 

Refrigerant Blends 

A. Laesecke (838) 

Thermal Conductivity of Natural Gas Constituents 

R.A. Perkins (838) 

Chemistry and Properties of Trace Components of Fuel Gas 

T.J. Bruno, W.C. Andersen (838); and A. Abdulagatov (Dagestan Scientific Center of 

Russian Academy of Sciences)

Stability of Potential High-Temperature Heat Transfer Fluids 

D.G. Friend, T.J. Bruno, and W.C. Andersen (838) 

 

Environmental Technologies 

Kinetics and Thermophysical Properties of Ionic Liquid Systems 

P. Neta, J.W. Magee, M.L. Frenkel, A. Laesecke, M.O. McLinden, R.A. Perkins (838); J. 

Grodkowski (Institute of Nuclear Chemistry and Technology, Warsaw); and G. Kabo 

(Belarusian State Univ.) 

Molecular Simulation of Solute Diffusion (Sodium Chloride) in Supercritical Steam 

A.H. Harvey and R.D. Mountain (838); and J.C. Bellows (Siemens Westinghouse) 

Industrial and Analytical Instruments and Services 

SpectroMLóan Extensible Markup Language for Molecular Spectroscopy Data 

A. Arslan, A.D. Nguyen, J.C. Travis, R. Sch‰fer, and G.W. Kramer (839) 

Microelectronics 

Thermophysical Properties of Gases Used in Semiconductor Processing 

J.J. Hurly, K.A. Gillis, and M.R. Moldover (836) 
144
145

14. Technologies for Future Measurements and Standards 

... anticipating and addressing next generation measurement needs of the nation 

CSTL maintains a strong basic research program in broad aspects of 

measurement science that positions us with the capability to provide the 

required measurements, standards, and data to support future technology 

development. New measurement techniques are important for a host of 

industries including biotechnology, healthcare, environmental technologies, 

and the chemical process industry. Many of our activities, such as protein 

crystallography and materials microanalysis, are at the leading edge of 

science and technology. 

Characterization of the Morphology of Faceted Nanoparticles by Transmission Electron 

Microscopy 

S. Turner and D.S. Bright (837) 

Characterization of the morphology of faceted nanoparticles is important in a variety of 

applications. The morphology controls the abundance, chemical composition and atomic 

arrangement of the particle surface and therefore controls the 

availability and activity of specific chemical sites on 

nanoparticles. For example, different crystallographic facets in 

catalysts have different efficiencies for promoting reactions. In 

pigment particles, certain morphologies are more desirable for 

the durability of paint. In the astrobiological field, considerable 

effort has gone into the 

characterization of faceted 

magnetite particles formed 

by some types of bacteria as markers for extraterrestrial life. 

Characterization of faceting by transmission electron 

microscopy (TEM) is challenging because a TEM image is a 

two-dimensional projection of material. In this work, the 

application of a double-tilt rotation holder to improved 

manipulation of particles was evaluated and a method for 

morphology modeling developed. By using this holder it is 

possible to orient a crystallographic direction of interest in a 

particle along a sample holder axis. A series of images is then 

collected and measurements of the dimensions made by using 

tools in a NIST-developed Lispix program. Possible models for 

the particle faceting are developed using commercially 

available SHAPE software. Comparisons of the experimental 

measurements to dimensions determined for different models 

of the morphology are then made to determine the best model 

for the particle 

morphology. 

Detailed knowledge of 

particle morphology can 

be critical to the chemical 

analysis of nanoparticles 

and to the increasing 

range of nanoparticle 

industry applications. 

Faceted rutile (TiO2) pigment 

particle oriented so that c axis 

(arrow) and projection of 

holder rod axis (dashed line) 

are aligned. Tilting around 

axis allows for improved 

morphology determination. 

200 nm 

[001]

This technique should allow chemical 

characterization of faceted particles to 

be more accurate because the x-ray and

absorption path lengths can be directly 

measured and modeled. 
146

Effects of Backscattered Electrons on the Lateral Resolution and Analysis Area of a 

Scanning Auger Microscope 

C.J. Powell, S.A. Wight, and J.T. Armstrong (837) 

The lateral resolution, δr, is a critical parameter in a scanning Auger microscope (SAM) because 

it is a measure of the distinguishability of small objects. For quantitative interpretations of Augerelectron signals, however, it is also necessary 

to consider the analysis area (the area from 

which all or a defined percentage of the 

detected signal originates). We have 

determined δr from linescans across a chemical 

edge for our newly acquired SAM at beam 

energies of 10 keV, 15 keV, 20 keV, and 25 keV. The magnification standard consisted of lines 

of chromium and chromium oxide of varying widths and separations on an indium-tin-oxide 

coating that was deposited on a fused 

silica substrate. The figure shows the 

fractional intensities of the In+O and 

Cr+O component Auger signals as the 

beam was scanned across an edge 

for each of the four beam energies. 

At each energy level, there is a rapid 

change in the Auger intensities in the 

vicinity of the edge and slow changes 

("tails") away from the edge. While 

these tails do not significantly affect the lateral 

resolution, they considerably increase the analysis 

area over simple expectations (e.g., that this area 

should be a small multiple of π(δr)

2

). This increase of 

the analysis area will significantly influence Auger 

analyses of particles and inclusions. 

Electronic Structure and Transport in Model Molecular Electronic Systems 

J.D. Batteas and S.W. Robey (837); R.D. van Zee and C.D. Zangmeister (836) 

The drive to increase electronic device performance, with the associated push to ever-smaller 

device dimensions, has lead industry observers to conclude that silicon-based technology will 

reach a point of diminishing gains in the near 

future. This, in turn, has generated interest in 

alternative technologies based, for instance, on 

single-electron devices and molecular 

components. It is hoped that the tremendous 

flexibility available with organic synthetic 

chemistry and self-assembly techniques can be 

harnessed to produce non-linear devices 

The analysis area needs to be carefully 

considered in quantitative Auger 

microscopy, particularly for "spot" 

analyses of particles or small inclusions. 

Auger linescans showing the 

relative fractions of the In+O and 

Cr+O Auger signals as a function of 

scan distance across a line edge of 

a magnification standard for beam 

energies of 10 keV, 15 keV, 20 keV, 

and 25 keV. 

These experiments and associated 

model calculations give guidance on 

the analysis area relevant to Auger 

analyses of particles and small 

inclusions.

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-3 -2 -1 0 1 2 3

In+O (10 keV)

Cr+O (10 keV)

In+O (15 keV)

Cr+O (15 keV)

In+O (20 keV)

Cr+O (20 keV)

In+O (25 keV)

Cr+O (25 keV)

Fractional Auger Intensity

25 keV

10 keV

25 keV

10 keV

Cr+O In+O

CSTL scientists are developing the 

measurement techniques and expertise 

necessary to understand electronic 

structure and dynamics in mesoscopic 

systems comprised of organic thin films 

and small molecular aggregates. 
147

analogous to silicon-based diodes and transistors, but comprised of single or small numbers of 

molecules. 

 The CSTL effort in molecular electronics includes 

developing a battery of techniques that will provide 

necessary information on electronic structure and 

electron transport in candidate molecular electronic 

systems. The methods being used include two-photon 

photoemission, which 

accesses unoccupied 

electronic levels and 

tracks electron relaxation 

effects, and scanned 

probed techniques, which 

can characterize electron 

transport down to the single molecule level. Validation of the 

performance of laboratory instrumentation against wellcharacterized systems is underway. 

An ultrahigh vacuum analysis 

system was assembled and coupled 

to a femtosecond Ti:sapphire laser for studies of electronic 

structure using two-photon photoemission. A scanned-probe 

system was purchased and preliminary studies of d-c transport 

through small numbers of conjugated molecules were also begun. 

Spectroscopic Study of the Structure of Oligo-Phenylene-Ethynlyene Monolayers with 

Molecular Electronics Implications 

C.S.-C. Yang, P.T. Wilson, and L.J. Richter (837); R. D. Van Zee (836); and J. J. Stapleton 

and D. L. Allara (Pennsylvania State University) 

Intense interest has recently been focused on the possibility of using chemical self assembly of 

appropriately chosen molecules to build electronic devices in a bottom-up fashion. This could 

lead to improvements in cost and device density over traditional semiconductor fabrication 

techniques. Simple electrical behavior such as 

conductance, rectification, and negative differential 

resistance (NDR) has been demonstrated in devices 

with a single monolayer of active molecules. Insights 

into the mechanisms underlying the observed 

molecular electronics phenomena have been limited 

due to the complete absence of spectroscopic tools appropriate to the study of the small 

molecular contacts. 

This work will provide information that, when coupled with 

theoretical input, will help to illuminate the physical 

mechanisms that produce the useful, non-linear effects 

observed in molecular electronics device-prototypes. This 

information is also required to develop measurement 

standards and test protocols for such devices. 

 

hν1

hν

2

e

-

Substrate Molecular

levels

EF

Evac

Two-Photon Photoemission

e

- 

AFM

Tip V 

Scanned Probe Transport 

The possibility of using electrically 

active molecules to build electronic 

devices has generated tremendous 

interest. 
148

CSTL, in collaboration with PSU, is exploring the potential of using advanced nonlinear optical 

probes to characterize the structure of electrically active molecular films. High quality monolayers 

were (molecule 1 and 2) formed on Au substrates at PSU. 

Vibrationally resolved sum-frequency generation (VR-SFG) 

spectra were recorded over the spectral range 1000 cm-1 to 

3200 cm-1 on both molecules at NIST. Shown in the figure 

are spectra recorded for two combinations of the 

polarizations of the three optical beams: ppp and ssp. The 

features at ≈1500 cm-1 and 

1600 cm-1 for both molecules 

are due to vibrations of the 3 

phenyl rings. The feature at 

≈1540 cm-1 in molecule 2 is 

due to the NO2 group. Also 

shown are predicted spectra 

based on ab initio calculations 

of the nonlinear line strengths 

of the two molecules. The ab initio spectra are in good 

agreement with the experimental spectra. The ratios of the 

resonance features for the two conditions can be used to 

determine the orientation of the molecule. 

Negative Differential Resistance Explained: Guidelines and a Model to Design New 

Molecules 

C.A. Gonzalez (838); Y. Simón-Manso (Universidad de Chile); and Y. Aray (Instituto 

Venezolano de Investigaciones Científicas)

The observation of abrupt changes in the slope in the 

current-voltage (I-V) characteristics of molecular devices 

formed by molecule-metal interfaces is one of the most 

promising phenomena in the field of molecular 

electronics. This phenomena could potentially lead to 

the manufacture of molecular electronic devices at the 

nanoscale such as diodes, gates, etc. Although there 

has been significant progress in the synthesis of such 

systems, the mechanisms leading to this behavior are not well understood. In this work a simple 

quantum-chemical model that provides insight into the understanding of the observed negative 

differential resistance (NDR) in current-voltage 

characteristics of molecule-metal tunneling 

junctions was developed and validated. The model 

is based in quantum chemical calculations of the 

electronic structure of bridge molecules in a 

capacitor-like electric field that mimics the potential 

spatial profile of the junction. 

VR-SFG spectra of 

oligo-phenyl-ethynlyene 

monolayers 

The tools used here have adequate 

spatial and temporal resolution to 

enable characterization of electronically 

perturbed states of the monolayer films.

Scientists from different microelectronics 

companies have indicated their interest in 

simple models like the one presented in 

this work, because it would help them 

“screen” a potentially large number of 

candidate molecules. 

CSTL is developing theoretical 

tools that can complement 

experimental efforts leading to 

the rational design of molecular 

electronics systems. 
149

The results show that a plausible mechanism for NDR behavior in planar molecular bridges, like 

the prototype molecule π-conjugated phenyl-ethylene oligomer 2í-amino-4,4í-di(ethynylphenyl)-5ínitro-1-benzenethiolate (PEO) involves a substantial rearrangement of the charge density of the 

neutral bridge at a threshold voltage. These changes are highly affected by the presence of the 

nitro group, which acts as an electron basin of attraction 

that forces the system to behave as a quantum well 

trapped between two barriers. Predictions based on the 

model indicate that substituting the nitro group for 

electron withdrawing groups such as fluorine atoms will 

also lead to NDR behavior. Currently, these predictions 

are being validated by measurements performed on the 

recently synthesized π-conjugated phenyl-ethylene oligomer 2í-amino-4,4í-di(ethynylphenyl)-5ífluoro-1-benzenethiolate. The model developed in this work provides a simple and powerful tool 

for the rational design of other molecular systems that could exhibit similar NDR behavior. 

Fabrication of the Building Blocks of Multifunctional Nanoscale Sensors 

R.M. Hernández and S.J. Stranick (837) 

Nanowire systems can provide multiple options for sensor 

design with additional sampling and measurement capabilities 

made possible by the miniaturization and integration of these 

components. Because any biological elements can theoretically 

interface with any of a wide variety of signal transducers (e.g., 

optical, electrochemical, 

thermal, or acoustic), the 

potential range of devices 

and techniques can be 

substantial. Nanowires 

composed of metal and 

conducting polymer segments were successfully fabricated 

using chemical and electrochemical synthesis inside the 

preformed nanoscale pores of anodic alumina membranes. The 

figure shows a 300 nm diameter, 5 μm long nanowire composed 

of alternating gold (bright) and polypyrrole (PPY) (dark) 

segments. Biochemical selectivity is imparted to the PPY 

segment by physical entrapment of antibodies, enzymes, and 

other receptors during 

electrochemical synthesis. In 

this work, the strong binding 

affinity between avidin and 

biotin was used to demonstrate biochemical selectivity as well 

as to access the impact of synthesis conditions on biological 

activity. In this experiment, the presence of entrapped, 

fluorescently labeled avidin in PPY was verified by 

fluorescence microscopy, and it was found that a PPY matrix 

can support electrical transduction without causing a 

denaturing of avidin or a loss of bioactivity in avidin: a 

necessary condition for the generation of conductance-based sensors. 

Optical micrograph of a 

300 nm diameter nanowire comprised of Au 

outer segments and a 

polypyrrole inner segment. 

Scale bar is 1μm. 

Nanoparticle and nanowire 

technologies are potential 

building blocks for nextgeneration analytical sensors. 

Nanowires with integrated 

chemical and biochemical species 

have been fabricated with the goal 

of having these structures serve 

as the building blocks in multifunctional nanosensors. 

A clear advantage of this 

entrapment process is that 

direct coupling or binding of 

the chemical/biological 

species is not required for 

immobilization/incorporation, 

i.e. a diverse array of chemical 

and biological analytes can be 

incorporated based on their 

ability to sense and rather 

than on their ability to bind. 
150

The Microanalytical Laboratory

L.E. Locascio, E.A. Waddell, T. Johnson, A.C. Henry, and P. Howell (839); D.J. Ross (836); 

and M. Gaitan (EEEL) 

Industries involved in the development and commercialization of microfluidic technologies are 

interested in polymer substrates to reduce microfabrication and materials costs. An important 

part of the NIST program is to disseminate 

microfluidic measurement methods, fundamental 

flow and surface characterization data, and 

surface modification protocols to facilitate the 

commercial development of polymer microfluidic 

devices. The goals of the Microanalytical 

Laboratory project are three-fold: (1) to establish 

a fundamental understanding of microfluid flow 

and develop models and methods to measure flow; (2) to devise methods for achieving chemical 

selectivity; and (3) to develop novel approaches 

for device integration. We have continued to 

focus a great deal of effort on understanding and 

controlling electroosmotic flow in polymer 

microfluidic systems. As a part of this work, we 

have studied the effect of polymer surface 

modification on microflow; developed methods for 

accurately measuring microflow; and developed a 

series of highly efficient passive micromixers and 

flow splitters that are easily integrated into 

polymer microchannels. We have recently forged 

a collaboration with CFD Research Corporation, 

an established fluid modeling company, to 

develop models of microflow in polymer 

microchannels that rely on NIST data. This year, 

we have also been successful in developing 

several novel methods for improved chemical 

separations in polymer microfluidic systems 

including temperature gradient focusing, and 

electro-chromatography with molecularly imprinted stationary phases. The advantages 

associated with both techniques are that they afford greater selectivity than other methods that 

have previously been implemented in a microfluidic format. This work continues to involve 

several active collaborations both internally 

(EEEL, PL, CSTL, and MSEL) and externally 

(Virginia Polytechnic and State University, 

Cornell, Stanford, Clemson, CFD Research 

Corporation). 

NIST scientists are targeting improved 

device performance as well as 

enhanced quality and reliability of 

chemical measurements performed in 

lab-on-a-chip devices. 

Experimental and simulated data on 

mass flow rate versus electric field 

strength for non-modified polystyrene 

channel (pristine PS) and chemically 

modified channel (COOH-PS).

NIST scientists have provided methods and 

data to support the growing microfluidics 

industry. A number of key collaborations have 

been established for the propagation of 

fundamental data and measurement methods. 
151

High Speed Elemental Mapping with the Silicon Drift X-Ray Detector (SDD) on an SEM 

D.E. Newbury, J.A. Small, and D.S. Bright (837) 

The silicon drift detector (SDD) is a new type 

of semiconductor energy dispersive x-ray 

spectrometer (EDS) capable of operating at 

much higher count rates than the 

conventional monolithic Si-EDS (at least 250 

kHz instead of 25 kHz). Spectrum imaging 

(SI), a powerful advance on conventional xray mapping, has been successfully 

demonstrated with the high-speed data 

stream of the SDD. In the SI mode, the beam is addressed to dwell for a specified time at a 

picture element (pixel) and a complete 

2048 channel EDS spectrum is 

recorded, thus creating a database array 

[m x n x I(E)] of m rows by n columns by 

intensity vs. photon energy [I(E), 2048 

channels]. The intensity per channel 

I(E) of this spectrum can be recorded to 

a depth of 16 bits. Because of the 

increased SDD photon counting speed, 

rapid mapping can proceed with a pixel 

dwell time as short as 10 ms, giving a 

total mapping time of approximately 650 

s for a 256x256 map. The SI mode 

permits the analyst to efficiently collect 

all the available spectral data with no 

loss and to subsequently interrogate the 

resulting data cube concerning any 

possible constituent of interest, even if 

questions arise long after the specimen 

has been removed from the SEM. 

Rigorous quantitative mapping 

procedures can be applied that make 

accurate background and peak deconvolution corrections to provide robust measures of minor 

and trace constituents, which are often misleading with conventional mapping procedures that 

rely only on ìspectrum windows.î The figure shows an 

example of a compositional map of the microstructure 

Raney nickel, a methanation catalyst, with the recorded 

database spectra obtained from a single pixel (pixel 

dwell time 10 ms) and from a 10x10 square of pixels. 

Major constituents are readily visible in the single pixel 

spectrum, while the 10x10 square of pixels has sufficient 

counting statistics for minor and trace constituents. 

Most mixtures of two or more elemental 

species tend to separate into two or more 

compositionally distinct phases at the 

micrometer to nanometer spatial scale. 

Existing x-ray mapping techniques are 

severely constrained by the count rate 

limitations of conventional Si-EDS. 

Raney Nickel

Ni-Al

E0 = 15 keV

256x256

10 ms

40 kcps

EDS Σ100 pixels

Intermediate phase

NiKα

NiKβ

NiL

AlK

Raney Nickel

Ni-Al

E0 = 15 keV

256x256

10 ms

40 kcps

EDS Single pixel

Intermediate phase

NiKα

NiKβ

NiL

AlK

Raney Nickel

Ni-Al

E0 = 15 keV

256x256

10 ms

40 kcps

EDS Σ100 pixels

Intermediate phase

NiKα

NiKβ

NiL

AlK Raney Nickel

Ni-Al

E0 = 15 keV

256x256

10 ms

40 kcps

EDS Σ100 pixels

Intermediate phase

NiKα

NiKβ

NiL

AlK

Raney Nickel

Ni-Al

E0 = 15 keV

256x256

10 ms

40 kcps

EDS Single pixel

Intermediate phase

NiKα

NiKβ

NiL

AlK Raney Nickel

Ni-Al

E0 = 15 keV

256x256

10 ms

40 kcps

EDS Single pixel

Intermediate phase

NiKα

NiKβ

NiL

AlK

Real time x-ray mapping will 

significantly improve this critical 

characterization technique with 

broad applications in materials 

science. 
152

Image series, bFd = 1.0-1.6, 

10% actual size. 

Images of Particle-Like Fractals with Differing Shapes and Boundary Fractal Dimensions 

for Testing Algorithms that Measure Fractal Dimension 

D.S. Bright (837) 

The shape of a single particle may indicate its 

treatment, chemical composition, or place of origin. 

Microscopists continually seek numerical parameters 

that quantify shape in order to characterize and 

classify particles. The boundary fractal dimension, 

bFd, is a parameter that describes the roughness of 

the outline of the silhouette of an object. Investigators 

have started to use the bFd in geology, tribology, 

pharmacology, medical research, aerosol science, and 

food science. However, the bFd values from objects in 

digital images differ, depending on the measurement 

method, and hinder broader acceptance of the bFd. 

Investigators have tested their methods only on a few well 

known fractals, and these fractals sometimes even have 

dimensions outside the range of the dimensions measured for 

their samples. Sets of images were made available covering a 

wide range of bFd values that were generated from a family 

of fractals related to the 

Von Koch snowflake. 

This family covers the 

full range of bFd, 1.0 to 

2.0, and has a range of 

shapes for each bFd. 

Not all of the fractals 

give suitable particlelike fractal images. 

Suitable images, their 

bFd values, and the 

software used to make any fractals from the family and 

measure bFd from their images are available on the web. 

The images and bFd values will 

help investigators to 

characterize the accuracy of 

their fractal dimension 

measurement methods so that 

results can be compared and 

reliably used. This should 

promote use of and confidence 

in bFd measurements to 

characterize particles. 

CSTL scientist provides a set of standard 

reference images and algorithms for testing and 

developing fractal dimension analysis for 

imaging systems. 

http://www.nist.gov/lispix/Lx04Doc/img-sets/Koch-fractal/koch-dnld.html
153

NIST Microcalorimeter Detector for High-Resolution X-Ray Detection 

J.A. Small, D.E. Newbury, R.L. King, and T. Jach (837); S.W. Nam, K.D. Irwin, and S. Deiker 

(EEEL) 

A NIST microcalorimeter energy dispersive x-ray spectrometer (μcal EDS) has been successfully 

transferred from NIST Boulder to an analytical microscopy environment at NIST Gaithersburg, 

along with routine operation and maintenance of the 

cryogenic and cryoelectronic systems. Initial 

measurements demonstrated resolution of 9 eV or better 

(at MnKα and 40% deadtime) for short duration 

accumulations (100 s), suitable for measurements of 

major (concentration, C > 0.1 mass fraction) and minor 

(0.01 < C < 0.1 mass fraction) constituents. Application 

of the μcal-EDS to resolving analytical situations with 

severe peak overlap in conventional EDS spectrometry (e.g., Cu-L and Zn-L) was demonstrated. 

Long duration (minimum 1000 s) measurements, necessary to accurately measure trace 

constituents (C < 0.01 mass fraction), were found to be subject to peak position and gain function 

instability. The instability results in 

degraded resolution by a factor of two 

or more. Researchers in Gaithersburg 

are currently working with NIST 

Boulder personnel to understand the 

cryoelectronic circuitry to diagnose and 

attain more stable energy calibration. 

The two overlapped spectra above demonstrate the 

stable (red spectrum) and unstable (black spectrum) 

regions on the same tantalum elemental standard. 

Future plans for the μcal-EDS x-ray 

spectrometer include an extensive 

program of fundamental 

measurements in support of 

microanalysis needs, such as the 

application of low-voltage 

microanalysis to nano-scale 

characterization, accurate values of 

the relative peak heights within x-ray 

families, peak shifts due to chemical 

bonding, as well as direct application 

to materials chemistry problems. 

NIST researchers are utilizing a 

micro-calorimeter energy 

dispersive x-ray spectrometer 

(μcal EDS) to support critical 

chemical measurement efforts 

for microanalysis research. 
154
155

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 four years 52 exploratory research 

projects have been funded, and CSTL has invested close to $4M. More importantly, we have 

been able to provide seed money for critical areas of research such as new approaches to 

modeling, measurement methods, or data development and dissemination, as well as novel 

mechanisms to provide reliable and timely measurement traceability to our customers. The work 

described in the following pages is a result of the research from FY 2002 funded projects. 

Development of Synthetic Protein-DNA Nanostructures 

D. T. Gallagher (831) 

CSTL Program Area: Biomaterials 

The design and construction of a molecular lattice 

is an active goal in many laboratories, with a variety 

of different motivating applications. Most potential 

uses for such lattices are as scaffolds for attaching 

functional molecules such as enzymes (to make 

bioreactors), electrical/optical components (to make 

information storage and signal processing devices), 

or proteins (to make crystals for diffraction-based 

structure determination). Although many advances 

in control of molecular assemblies have been 

reported in recent years, no one has yet produced 

a designed, regular molecular lattice. 

Choice of Materials: We have chosen to use 

protein and DNA to build the lattice. DNA is useful 

because it has known and predictable structural 

properties, forming (as duplex) long straight helical 

rods. Protein is necessary because DNA alone 

does not branch or crosslink; proteins that are 

already known to bind DNA will serve as lattice 

nodes. Of the approximately 200 DNA-binding 

proteins of known structure, several have geometry 

that is potentially suitable for lattice assembly. We 

selected the protein represented by Protein Data 

Bank structure 1AN2 for initial studies. This is a 

human transcription factor of the leucine zipper 

type, which binds DNA as a symmetric dimer. It 

was necessary to truncate the protein in order to be 

compatible with the lattice, so we will use only the 

first 51 amino acids, which includes both the DNAbinding and dimerization functionalities. 

 

Design: A detailed (atomic) structural model has been developed for one design of a proteinDNA nanolattice. This lattice design has I4122 symmetry and about 15 nm repeat spacings. The 

Some see the creation of a molecular 

lattice as a goal in itself, since it is 

consistent with the general nanotechnology trend toward finer 

structural control. At NIST, such 

lattices, by virtue of their precisely 

known geometry, will enable the 

development of powerful new 

measurement methods for biomolecular materials. 

The difficulty is that lattice subunits must 

be capable of assembling in only one 

way. This implies that the subunits must 

be rigid and homogeneous, and that their 

geometry dictates specific controlled 

polymerization (lattice assembly) in three 

dimensions. Traditional organic 

polymers are either too flexible, too 

difficult to produce as homogeneous 

species, or lack the geometric properties 

that could support 3-D lattice assembly. 

Inorganic approaches such as metal 

coordination may produce crystals, but 

with spacings too small to support guest 

functionalities. 
156

protein component is a disulfide doubled form of the dimeric (and truncated) 1AN2 protein. The 

added cysteine residues are at the N-terminus, a position that gives the resulting dimer-of-dimers 

the required 90-degree dihedral angle between the two bound DNA duplexes. The duplexes will 

then be given appropriate length (odd number of turns, divided by four) and termini (two-base 

sticky end overhangs) to enable assembly with other 

subunits. 

PCR (polymerase chain reaction): We are currently 

using PCR methods to assemble a synthetic gene for 

the engineered protein, so that protein expression and 

purification can lead to assembly (with separately 

synthesized DNA components) of the lattice. Initially 

we will produce the protein without the terminal 

cysteine, so that it will bind only one DNA duplex. This 

can be easily verified by polyacrylamide gel 

electrophoresis methods. Once the DNA binding 

property is confirmed, then the cysteine form will be 

produced, and its disulfide dimerization assayed. 

Next Steps: The cysteine-free engineered protein is predicted to retain both its dimerization and 

DNA-binding properties, but this must be verified. Following that, the cysteine form will be 

expressed and tested for formation of the dimer-of-dimers. The dimer of dimers will then serve as 

the bivalent module as it will bind two different DNA duplex molecules. 

Production of Soluble and Enzymatically Active Gene Products (Proteins) in Escherichia 

Coli

P. Reddy (831) 

CSTL Program Area: Pharmaceuticals and Biomanufacturing

In the protein expression endeavors, we frequently experience the production of inactive and 

insoluble protein aggregates called 

"inclusion bodies" or no protein expression 

at all due to proteolytic degradation. The 

framework of this project is directed towards 

engineering a universal protein expression 

plasmid vector that should provide a 

solution to these frequently encountered 

problem in protein expression. 

We used the recombinant expression plasmid (pRE) vector with 

tightly controlled transcription/translation signals, which was 

originally developed for cloning and overproduction of lethal 

proteins. We then fused a segment of the gene for a chaperone 

protein GroEL, which is a universal protein folding machinery in 

the cell. A short DNA molecule that codes for a peptide linker 

with a specific protease (enterokinase) recognition sequence for 

subsequent release of the protein and a unique restriction 

endonuclease recognition sequence (CATATG) were 

incorporated downstream of the GroEL gene in the vector. All 

the fusions are in-frame with initiator ATG. The strategy is that 

the improved plasmid vector could then be used for cloning and 

overproduction of any gene. 

CSTL researchers are aiming to 

produce a bivalent DNA-binding 

protein module that can serve in the 

assembly of specific biomolecular 

nanostructures. With appropriately 

designed DNA components, various 

constructs of increasing complexity 

(1-D, 2-D, 3-D, single subunit, two 

subunits, etc.) can be produced by 

small modifications in the design, 

providing a testable route to the 

higher complexity of the extended 

3-D lattice. 

Protein expression/gene expression is of 

vital importance to the biotechnology/ 

pharmaceutical industry to overproduce 

proteins in order to design small molecular 

drugs and protein pharmaceuticals and to 

fully understand cellular function. 
157

One test gene (Mycobacterium smegmatis calmodulin) was chosen for making fusion to GroEL in 

the newly constructed plasmid vector. The test gene product was not expressed at all by itself 

perhaps due to proteolytic cleavage in the cell. Upon expression of the GroEL:calmodulin gene 

fusion in E. coli, we observed that calmodulin gene was successfully expressed as a fusion 

protein. We subsequently purified the fusion protein, cleaved the fusion protein with enterokinase 

and released calmodulin from GroEL. Hence, in principle we have constructed a plasmid vector 

for expression of soluble proteins. 

Genome sequencing of several microbes and eukaryotes has revealed a wealth of knowledge on 

the gene segments that code for proteins. Nearly 50% of the proteins have no known function 

and hence are termed “hypothetical proteins”. The challenges in the post-genomic era are:

cloning of hypothetical genes, expression of the corresponding gene product (protein), and 

characterization of the protein for its function. 

Next Steps: The vector will be tested with a variety of genes that are known to produce inclusion 

bodies. 

Rapid Analysis of the Kinetics of Enzymatic Reactions by a Novel Stopped-Flow 

Microcalorimetry Method 

F. P. Schwarz (831/CARB); and M. Stodeman (CARB/UMBI) 

CSTL Program Area: Pharmaceuticals and Biomanufacturing

Research in microcalorimetry has lead to the development of computer-controlled, highthroughput, rapid time-response and highly sensitive differential stopped-flow microcalorimeters 

(DSFM) that are amenable to monitoring the progress of an enzyme-substrate reaction 

calorimetrically. DSFM data for two types of enzymesubstrate reactions were analyzed in terms of the 

integrated form of the Michaelis-Menten rate 

equation to yield values for Km, the substrate 

concentration required for the reaction to proceed at 

half its maximum velocity, and kcat, the reaction 

turnover number. Analysis of the isomerization of 

fructose-6-phosphate to glucose-6-phosphate by 

phosphoglucose isomerase yielded values for Km and 

kcat from 293.4 K to 311.5 K and values for the 

product-substrate equilibrium constant that agreed 

with literature values. Analysis of the hydrolysis 

reaction of four N-acetyl amino acid derivatives by 

acylase at 298.2 K yielded values for Km and kcat, that 

were in fair agreement with values obtained by other methods. The exothermic heats of reaction 

for each substrate-enzyme reaction were also obtained at each temperature and exhibited an 

increase with temperature. 

The isomerization of fructose-6-phosphate to glucose-6-

phosphate by phosphoglucose isomerase exhibits significant 

substrate-product equilibration. This exothermic reaction was 

monitored at 293.4 K, 298.4 K, 303.4 K, and 311.4 K and 

reversibly (endothermically) at 293.4 K and 298.4 K. Values 

of the equilibrium constant determined from the reverse and 

forward reactions were in agreement with literature values. 

The hydrolysis of N-acetyl derivatives of L-methionine, 

glycine, phenylalanine, and alanine at 293.2 K by acylase is 

exothermic and exhibits significant acetate product inhibition. 

Since virtually all of the enzymesubstrate reactions involve the 

exchange of heat with the 

surroundings, the progress of any 

enzyme-substrate reaction can be 

universally and efficiently monitored

by a DSFM. Rapid evaluation of 

enzyme kinetic parameters and their 

dependence on temperature and pH 

are needed by the biocatalyst 

industry and are needed to identify 

the function of unknown proteins. 

Major Accomplishments: 

More than 80% of the 

progress of the hydrolysis 

and isomerization reactions 

monitored by DSFM agreed 

with the analytical 

expressions derived from the 

intergrated form of the 

Michaelis-Menten rate 

equation. 
158

The hydrolysis of N-acetyl-L-methionine was also investigated at 288.4 K, 308.5 K, and 318.5 K. 

Values for Km and kcat at 293.2 K were within the wide range of literature values determined by 

the more conventional reciprocal velocity versus substrate concentration plots. The results of the 

acylase investigation were presented at the 57th Calorimetry Conference and in a publication that 

is in press in Analytical Biochemistry. 

Next Steps: A paper on the acylase reaction was submitted to WERB and a third one on the 

isomerization reaction is in preparation. Plans are underway to examine other types of enzyme 

reactions by DSFM. 

Localization of Boron Neutron Capture Chemotherapeutic Agents by Secondary 

Ion Mass Spectrometry 

S.V. Roberson and G.J .Gillen (837); and S. Chandra (Cornell Univ.) 

CSTL Program Area: Health and Medical Technologies 

This project evaluated the use of secondary ion mass spectrometry (SIMS) to image the location 

of isotopically tagged boronated compounds in T98G human glioblastoma cell cultures and to 

evaluate cell viability by means of the Na/K ratios. We have mapped the distributions of 10B, Na, 

Mg, K, and Ca in freeze-fractured cell 

cultures. This work has the potential to 

provide valuable information regarding the 

optimization of therapeutically useful 

dosages for 10B-containing compounds. 

Future plans include localizing the 

chemotherapeutic agent by means of 

molecular secondary ion signals and 

exploring means to enhance the signals 

obtained. 

The purpose of this project was to use the high spatial resolution isotopic and molecular imaging 

capabilities of secondary ion mass spectrometry (SIMS) for the localization of isotopically tagged 

boronated compounds in tumor cell tissue cultures. There is significant interest in utilizing boron 

neutron capture therapy (BNCT) as a form of cancer radiation treatment that destroys cancer cells 

without injury to adjacent healthy tissue. BNCT has demonstrated effectiveness for treatment of 

several forms of cancer including glioblastoma and melanoma. Due to a lack of analytical 

techniques capable of measuring in-vivo 10B concentrations (typically less than 100 μg/g by mass) 

in tumors at the time of treatment, optimizing the therapeutic regimes has proven difficult. SIMS 

imaging is an ideal candidate for evaluating the distribution of BNCT drugs in tumor cell cultures 

and was used in this work to map Na, K, Mg, and Ca distributions as well as the 10B and 11B 

isotopes at sub-cellular spatial resolution. 

One of the primary issues addressed in this work was the viability of the cell freeze-fracture 

preparation process and development of a method to identify undamaged cells. Cell cultures on 

silicon are rapidly frozen in liquid nitrogen to maintain the in-vivo distribution of diffusible elements 

and the chemotherapeutic agent. If the cell is injured in this process, the plasma membrane 

becomes permeable to ion gradients. Cells will consequently lose intracellular potassium and 

gain extra-cellular sodium. Cell damage occurs frequently during the freezing process. This 

damage can invalidate the SIMS imaging experiments due to possible migration of the analyte 

molecules. It is critical that regions of undamaged cells be localized for analysis. We use the Na 

and K secondary ion image intensities as indicators of cell viability. The image shows secondary 

ion images from a freeze-fractured specimen of a T98G human glioblastoma cell culture, 

containing 10B enriched boronophenylalanine (BPA). A 25 keV Ga+

 primary ion was rastered over 

a 111 μm x 111 μm area to acquire the images. Damaged cells show high levels of sodium and 

Glioblastoma multiforme is the most 

malignant form of brain cancer. Boron 

Neutron Capture Therapy (BNCT) has proven 

to be an effective treatment option for this 

form of cancer and provides a mechanism to 

destroy cancerous cells without injuring 

neighboring normal cells. The success of 

BNCT relies on the selective loading of 

boron in the tumor cells. 
159

low levels potassium. The images show expected ratios for a region containing healthy cells. A 

second major issue was to determine if sub-micrometer localization of the 10B were possible at 

physiologically relevant concentration levels. 

The figure also shows the 10B distribution in the cells. Sufficient sensitivity is 

available to determine the distribution of the 10B. 10B appears to distributed 

nearly homogenously throughout the cells. We deduce that the more 

intense signals (brighter areas) that are seen on the periphery of the cells 

are due to an artifact of the SIMS experiment and do not reflect an actual 

concentration gradient. The origin of this artifact will be addressed in future 

work. Because of the low concentration of the boronophenylalanine, 

secondary ion counts for the 10B are one to two orders of magnitude lower 

than for the matrix elements, Na and K. In order to increase the sensitivity 

of the analysis, further analyses are being conducted using an O2

+

microbeam. A collaboration has also been initiated to develop a new highbrightness ion source for this application. 

Next Steps: This preliminary work 

provided promising data on the 

feasibility of utilizing SIMS to 

determine the localization of 

boronated cancer chemotherapy 

drugs. In the future, we will 

concentrate our efforts on increasing 

elemental secondary ion signals. We 

are collaborating with a small 

company on the design of a highbrightness O2

+

 source for microbeam 

imaging on our magnetic sector instruments. Additional plans include 

mapping molecular secondary ion signals, locating sub-cellular structures, 

and quantifying the chemotherapeutic agents. 

Development of Methods for the Speciation of Selenium in Health Markers 

S.E. Long, D,M. Bunk, M.A. Arce-Osuna, and S. Christopher (839) 

CSTL Program Area: Health and Medical Technologies 

Selenium deficiency is correlated with damage to cell membranes due to accumulation of free 

radicals, while it also appears to be a pathogenic factor in the onset of osteoarthropathy, 

cardiomyopathy, and muscular disorders. Prior studies have been limited by large variability in 

the reported analytical data and difficulties associated with the absolute identification of the 

selenium species. Conclusive information on selenium 

content and metabolism in the human body has 

therefore been limited. The purpose of this work has 

been to develop an analytical measurement system for 

the separation and positive identification of relevant 

seleno-proteins and to accurately quantify them using 

inductively coupled plasma-mass spectrometry (ICPMS). A new patentable hydride generation cell design 

together with a flow control system have been 

developed, which may be used to accurately quantify 

extremely low levels (pg/g) of selenium by ICP-MS. 

The success of BNCT relies on 

the selective loading of boron in 

the tumor cells. The results 

indicate that SIMS is ideally 

suited for assessing cell health 

and for the evaluation of BNCT 

drugs. The technique could be 

used to provide initial screening 

and evaluation of the efficacy of 

new boron therapeutic agents. 

The role of selenium metalloproteins in the body is a subject 

of increasing interest to the 

clinical community. The 

development of a more accurate 

measurement system within 

CSTL may be used to provide a 

traceability link that would 

benefit the clinical community.
160

The goal of this research is the development and implementation of new analytical procedures for 

the positive identification and accurate quantitation of selenium species in targeted clinical 

systems. Previous studies have often been limited by poor analytical sensitivity, species 

specificity, contamination and losses that have led to conflicting information on reference intervals 

and estimates of toxicity and deficiency status. The 

new device developed by these researchers is 

based on a permeable membrane separation 

system, which can be mounted directly onto the 

ICP torch assembly. The design can be used to 

measure extremely low concentrations of 

selenium. The accurate determination of selected 

selenium species in clinical systems and the 

provision of reference systems will provide 

traceability for measurements by the health-care 

industry and for the in-vitro diagnostic (IVD) 

market. 

Next Steps: This work will be assumed for a longer term by a recently arrived Ph.D. student. The 

isolation of seleno-proteins from serum will be accomplished using a multidimensional 

chromatographic approach, using metal affinity chromatography to selectively extract seleniumbound proteins from an abundance of serum proteins. Additional chromatographic steps will be 

used to further separate the seleno-proteins from each other before characterization by mass 

spectrometry of the intact protein and after proteolytic fragmentation. Amino acid analysis will be 

used to determine the presence of selenomethionine, selenocysteine, or selenocystine amino 

acid residues. Once the serum seleno-proteins have been identified, quantification of their 

selenium content can be made by LC/ICP-MS. 

XML for Microanalysis 

J. H.J. Scott (837) 

CSTL Program Area: Industrial and Analytical Instruments and Services

XML development efforts on this project focused carefully on the original scope of the project 

(creating a spectrum file format), but the eventual XML application was designed to integrate with 

future components such as sample information file formats and instrument information file formats 

without sacrificing its performance as a spectral exchange tool. After testing several variations of 

the markup language and exploring design 

tradeoffs, a working prototype was implemented 

to store test data. 

Although the original goal of this work was to 

provide a standard file format for the exchange of 

spectral data between microanalysis laboratories, 

it quickly became clear that this technology could 

provide a framework for broader information 

sharing in the microanalysis community. A welldesigned Microanalysis Markup Language (MML) 

can capture information about all aspects of a 

microanalysis experiment (not just spectral 

results). This has led to a natural convergence of this project and the SpectroML project led by 

Gary Kramer (additional information provided in the Industrial and Analytical Instruments and 

Services section). Although the two formats remain distinct because of the specific information 

being represented, the inherent flexibility of XML ensures compatibility and interoperability 

between the two applications. Because XML-formatted data exchange in the x-ray spectrometry 

industry is still in its infancy, an important opportunity has arisen to work on an open standard 

Major Accomplishments: 

1. Definition of critical measurement 

needs of the clinical community. 

Target clinical markers have been 

identified as total selenium, as well as 

selenoprotein P, albumin, and the 

seleno-amino acids in blood and 

urine. 

2. Development of a new hydridegeneration interface and flow control 

system for the sensitive determination

of selenium by ICP-MS. 

The purpose of this project is to enable 

the free exchange of spectral 

information among microanalytical 

instrument users by developing an 

XML-compliant markup language for 

spectroscopy data and to provide an 

information framework for storing and 

sharing microanalysis data communitywide, allowing more sophisticated 

theoretical model building and model 

assessment to take place. 
161

format with all the manufacturers before a proprietary approach has taken hold. One express 

purpose of this work is to encourage consensus among the end users and x-ray analysis system 

vendors. Buy-in from both the analytical instrument users and the system vendors is critical to the 

success of this project. 

After testing several variations of the markup language and exploring design tradeoffs, a working 

prototype of the Microanalysis Markup Language (MML) was implemented to store test data. 

Variations using XML attributes in place of some elements were considered, but these were 

abandoned in favor of a more normalized approach. 

Next Steps: Future work on the markup language will attempt to refine and improve the 

implementation, taking account of the 

suggestions from the user/vendor task 

group after circulation of a draft 

standard. A small group of beta test 

sites and working microanalysis 

laboratories will be used to ensure the 

XML application is practical and not 

burdensome. If sufficient industry 

interest is shown, NIST will aid 

industry in developing a national 

and/or international standard through 

ASTM E42 or ISO/TC202. 

In addition to sparking immediate interest among end 

users and industry representatives at Microscopy & 

Microanalysis 2002, this work led to the reactivation of 

the Microbeam Analysis Society’s computer activities 

subcommittee headed by Nestor Zaluzec at Argonne 

National Laboratory. This same committee helped 

develop the EMMFF file format, a simpler ASCII-based 

spectrum exchange format currently in use. XEDS 

manufacturers offered non-financial support and 

collaboration opportunities along with implementation 

suggestions and requests for features. 

<?xml version="1.0"?> 

<experiment> 

 <spectrum> 

 <title>S8 Cu 20kV 20nA UTW</title>

 <date>2002-08-07</date> 

 <time>11:45:00.0</time> 

 <owner>John Henry Scott</owner> 

 <xunits>keV</xunits> 

 <yunits>counts/s</yunits> 

 ¦ 

 <x-mm>0.0000</x-mm> 

 <y-mm>0.0000</y-mm> 

 <z-mm>0.0000</z-mm> 

 <edsdetector>SIUTW</edsdetector> 

 <spectrum-data> 

 <frame npoints="2048"> 

 0.000 0.0 

 0.010 3.0 

 0.020 1.0 

 0.030 6.0 

 0.040 5.0 

 0.050 8.0 

 0.060 20.0

Example Microanalysis Markup Language (MML) excerpts showing element 

structure (left) and default browsing display in Internet Explorer (right). 
162

Fluorescence Thermometry for Temperature Measurements of Microscopic Surfaces 

C. W. Meyer, K. G. Kreider, S. Semancik (836), and E. S. Etz (837) 

CSTL Program Area: Microelectronics

The objective of this research is to make use of the temperature dependence of the fluorescence 

lifetime of certain phosphors for temperature measurement on a microscopic scale. The purpose 

of this research is to determine the viability of 

fluorescence lifetime microscopy (FLM) for 

measuring temperatures of surface areas with 

length scales 1 μm to 200 μm. FLM has 

become a highly developed technology over 

the past decade and it has been used 

extensively for biomedical research. The 

temperature-dependence of the fluorescence 

lifetime of certain phosphors gives FLM the 

potential to be a powerful tool for temperature 

measurement on a microscopic scale, but up 

to this point it has never been used for this 

purpose. 

The phosphor to be used for the fluorescence thermometry (Mg4FGeO6:Mn) has been obtained 

and fabricated into targets for sputtering onto the substrates of interest. Films of 1 μm have been 

sputtered onto the calibration block for preliminary studies and calibration. The assembly of the 

fluorescence microscope has been completed by mounting on the microscope a pulsed blue LED 

(λ = 450 nm) for the excitation of fluorescence along with a photomultiplier tube (PMT) for 

detection of the fluorescence. An oscilloscope is currently being used to monitor the output of the 

PMT, and soon an analog-to-digital (A/D) converter and computer will be used to analyze the 

output of the PMT to determine the fluorescence lifetime. 

Next Steps: Once the preliminary tests of the apparatus are done and it is shown to work 

properly, we will calibrate the fluorescence lifetime of the phosphor against a calibrated contact 

thermometer. The results of microscopic measurements will be compared to those performed for 

macroscopic surface areas. Measurement reproducibility using different samples of the phosphor 

and the overall temperature measurement 

uncertainty will be determined. The spatial scales 

of the phosphor will be changed by changing 

microscope magnifications, and we will determine 

if FLM performs successfully and consistently for 

surface-temperature measurements at all available 

spatial scales. Assuming the FLM performs 

successfully, we will use it on microhotplates 

coated with a thin film of Mg4FGeO6:Mn for 

measuring temperature gradients and performing 

thermal imaging. 

This work is expected to fulfill a need in 

microelectronics applications for 

accurate surface temperature 

measurements to monitor heat 

dissipation in power devices, such as in 

bipolar junction transistors and 

MOSFETs. It may also help improve the 

performance of small chemical sensors 

and biosensors, where the signal outputs 

are often temperature dependent. 

The objective of this project is to use FLM 

for measuring temperatures and 

temperature gradients on microhotplates, 

satisfying the need for measurement of 

these quantities on microhotplates and 

also demonstrating the potential of this 

approach for all applications in need of 

such measurements. Potential 

beneficiaries include those who work with 

microelectronics, microscopic chemical 

sensors and biosensors, and MEMS. 
163

The Initiation Step in Pyrophoric Combustion of Silane 

K.K. Irikura (838) and M.S. Gordon, I. Adamovic, and H.M. Netzloff (Iowa State Univ.) 

CSTL Program Area: Data and Informatics

In the semiconductor industry, combustion of pyrophoric gases such as silane (SiH4) and 

phosphine (PH3) is an important safety hazard. Although the combustion can have explosive 

force, it is initiated by relatively slow reactions. After an induction period, the primary products 

react rapidly to produce the characteristic 

flame or explosion. Silane combustion has 

been the subject of significant kinetics 

modeling efforts at NIST and elsewhere, but 

the initial step remains conjectural. We have 

suggested some new mechanisms for this 

reaction and are evaluating them using 

advanced methods of quantum chemistry. In 

addition to its importance for pyrophoric 

combustion, similar chemistry may be 

responsible for initiating the autoxidation of 

organic compounds, including plastics, 

elastomers, lubricants, and edible fats and 

oils. An understanding of how these reactions 

occur may lead to new ways of suppressing 

undesirable oxidative reactions. 

Superficially, the pyrophoric combustion of semiconductor-processing gases might not appear to 

be related to autoxidation. Both processes, however, involve oxidation by atmospheric oxygen 

and chain reactions that follow a much slower, unknown initiation step. In this project, we chose 

silane combustion as a model system for autoxidation due to the following advantages: (1) silane 

is a well-defined compound, (2) experimental data on ignition delays are available for comparison 

with models, (3) kinetic models have already been developed, (4) it is a small molecule amenable 

to high-level theory, and (5) it is of practical interest in its own right. We consider initiation 

reactions involving singlet and triplet oxygen, and also the “two-state” mechanism discovered 

recently in the gas-phase oxidation of saturated 

hydrocarbons by FeO+

. 

Next Steps: This project is in its early stages. 

We intend to evaluate the alternative 

mechanisms over the coming year, which will 

include kinetics modeling elsewhere in CSTL. If 

this project is successful, future work will address 

the major, long-standing problem of hydrocarbon 

autoxidation. 

Oxidative degradation of hydrocarbon 

derivatives is the organic equivalent of 

rusting. Unwanted oxidation of plastics, of 

which the U.S. produces 100 billion pounds 

annually, presents an important economic 

burden. Likewise, lubricating fluids no 

longer function properly if oxidized. Edible 

fats and oils become rancid and unfit for 

consumption. These chemical processes 

are all characterized by slow initiation 

followed by a faster chain reaction. 

Despite the practical interest, most 

purported progress has been merely 

speculative. 

A contract was awarded to Iowa State 

University (ISU) in August. Nonetheless, 

we have already obtained a preliminary 

transition structure for the singlet reaction. 

The preferred theoretical method has also 

been identified. Although this capability is 

unavailable in standard software packages, 

it has been programmed into the 

developmental version of the ISU software. 
164

Bimolecular Reaction Rates and Mechanisms Relevant to Nano-structure Formation 

J.W. Hudgens (838)

CSTL Program Area: Data and Informatics

Gaseous organometallic compounds are used during manufacturing processes to deposit metals 

in semiconductor, optical, fuel cell, MEMS, and NEMS devices. The metal deposition rates and 

film quality are governed by the chemical environment above substrate surfaces formed by 

precursor compounds, by their decomposition 

products, and by the secondary products formed 

through bimolecular reactions with free radicals. 

Chemical modeling of any metal organic chemical 

vapor deposition (MOCVD) process is difficult due to 

the absence of reliable kinetic rate equations for 

organometallic species. Moreover, useful reference 

mass spectra are unavailable. This project comprises 

two laboratory activities. The first uses a novel "beamswallowing" mass spectrometer to measure the first 

reference mass spectra of organometallic compounds. 

During FY2002 this apparatus was designed and its 

components were procured. The apparatus uses a pulsed molecular beam valve to introduce 

organometallic molecules for analysis. The design uses a series of apertures to shape the 

molecular beam so that thermally labile species 

never contact a hot ionizer surface. Because 

this "beam-swallowing" geometry inhibits the 

formation of pyrolysis products, the apparatus 

can measure high-quality reference mass 

spectra of radicals and unstable chemical 

species. By mounting a small pulse-heated, 

high-pressure flow reactor to the molecular 

beam valve, fundamental bimolecular 

mechanisms and reaction rates will also be 

measured. 

These data will become available through the 

NIST Mass Spectral Database web site. The 

second uses mass spectrometry to measure bimolecular reactions between organometallic 

precursors and free radicals. The fundamental rate data will become available through the NIST 

Chemical Kinetics Database web site. 

Next Steps: Future plans include measurements of reference mass spectra and bimolecular 

reaction rates. Reference mass spectra of organometallic compounds will become available 

through the NIST Mass Spectral Database web site. Bimolecular reaction rate coefficients, 

initially for M(CH3)n (M= Sn, Cu, Ga, Zn) species, will become available through the NIST 

Chemical Kinetics Database web site. 

This project will provide reference 

mass spectra and fundamental 

chemical kinetic properties of 

organometallic compounds that 

will facilitate reactor design, 

improve process monitoring 

accuracy, and enhance the safety 

of processes used to fabricate 

semiconductor, optical, fuel cell, 

MEMS, and NEMS devices. 
165

Development of Accurate Quantitative Analysis Procedures for High-Angle X-Ray 

Emission Spectroscopy 

J. T. Armstrong and J. A. Small (837) 

CSTL Program Area: Technologies for Future Measurements and Standards

Electron backscatter diffraction (EBSD) is an important new analytical technique for micro- and 

nano-scale characterization of crystalline materials in scanning electron microscopes and similar 

instruments. There are several commercial 

manufacturers of EBSD detectors and this technique 

is being actively used for crystalline phase 

identification and phase orientation mapping of 

advanced materials with important commercial and 

defense applications. To properly characterize 

unknown materials, it is often critical to combine 

EBSD with quantitative x-ray emission (EDS/WDS) 

analysis; in most cases, the practical requirement is 

that these measurements be done simultaneously. 

However, EBSD analysis needs to be performed with 

the sample inclined so that the electron beam strikes 

it at a shallow angle (typically 20° above the surface plane); at such tilt angles, conventional 

correction procedures for x-ray analysis are not accurate and there are no generally agreed-upon 

alternatives. We have initiated work to determine the best analytical methodology, quality 

assurance testing, and data correction procedures to employ for high-tilt-angle EDS/WDS. 

The procedures and corrections explored were designed specifically to enable electron 

backscattered diffraction analysis (EBSD) and quantitative x-ray emission analysis (EDS/WDS) to 

be performed simultaneously on commercial scanning electron microscopes. They also enable 

integration of quantitative EDS/WDS with a variety of other surface and microanalysis techniques, 

such as Auger electron spectroscopy, x-ray photoelectron spectroscopy, and scanned probe 

microscopies. For the first time these procedures enable analysts to perform, simultaneously, 

accurate elemental analysis along with the best of the modern surface analysis methods. The 

beneficiaries of these procedures are microanalysts in a wide variety of commercial and 

homeland security applications from pharmaceuticals and microelectronics to explosives and 

forensic analyses. 

We have performed extensive Monte Carlo calculations of electron trajectories in solid targets 

inclined at various angles with respect to the electron beam. We have tested these with 

experimental measurements made on our analytical scanning Auger microprobe, which is 

specially configured for doing x-ray emission analysis of inclined specimens. Preliminary 

measurements were also made on our field-emission 

scanning electron microscope with EBSD. These 

two instruments, because of the different azimuthal 

angles at which the x-ray detectors are mounted, 

represent the extreme conditions under which EBSD 

measurements are made. The experimental results 

are in good agreement with the Monte Carlo 

calculations, but show relatively poor agreement to 

several empirical corrections that had been adapted 

for shallower beam tilt angles. The peak-tobackground ratio for x-ray lines with large 

overvoltage (U = E0/Ec > ≈3 to 4) at high tilt were 

found to be within 10% of the values measured at 

normal incidence, suggesting that the correction method for particles and rough surfaces 

proposed by Small could be employed for tilted samples as well under these conditions. 

A practical series of analytical and 

quality control procedures were 

developed and an evaluation of 

microprobe correction algorithms 

was made to provide a robust 

method to enable quantitative xray microanalysis to be performed 

at large, variable specimen tilt 

angles relative to the electron 

beam and x-ray detectors.

An easy-to-use internal standard quality 

assurance check was developed to 

ensure reproducible positioning. A 

polished specimen of SRM 482 AuCu is 

mounted with the sample. Using the 

AuCu standard, the ratio of the Au-L to 

Au-M and Au-M to Cu-K are measured. 

These ratios are very sensitive to beam, 

specimen, and detector geometry. 

Reproducing these ratios to a set value 

assures a consistent geometry, which 

enables Monte Carlo based corrections 

to be used to process analytical data. 
166

However, the measured peak-to-background ratio for higher energy lines (U < ≈ 3), at high 

specimen tilts, differs significantly from the values measured at normal incidence. The Monte 

Carlo simulation results indicate that polynomial “a-factors” (such as developed by Armstrong for 

normal incidence thick specimens and microparticles) could be tabulated and employed in a 

simple-to-use correction procedure. Demonstration matrices were calculated and found to 

produce accurate results. In performing the experimental measurements, we found that great 

care was needed to properly reproduce the correct sample and detector positions. The x-ray 

path length (and thus the amount of x-ray absorption vary significantly with detector azimuthal 

position, sample tilt (in both x and y directions) and sample z-positioning (height). The analytical 

results are highly sensitive to these factors. 

Development of analysis and correction procedures enabling quantitative EDS/WDS analysis at 

high specimen tilt angles answers several of the most critical current microanalysis needs of 

industry. It enables simultaneous quantitative x-ray emission and EBSD analysis and phase 

orientation mapping. The quality assurance approach allows microanalysts to consistently 

operate at defined analytical geometries. The results of this project also enable x-ray emission 

analysis to be performed simultaneously with other complementary techniques that typically have 

tilted samples such as Auger electron spectrometry, dual beam FIB (focused ion beam), x-ray 

photoelectron spectroscopy, and the various ion, laser, and scanned probe techniques. By not 

requiring the electron beam to be perpendicular to the sample surface, these techniques enable 

development of hybrid and miniaturized instruments that would be still capable of high-accuracy 

x-ray analysis. Application of this approach can aid industry in many areas including improved 

quality control, failure analysis, process control, device fabrication, polymer and biomaterials 

characterization, etc. 

Next Steps: This work completed a one-year feasibility study conducted as an exploratory 

research project. The results are promising enough to warrant future work. We plan to distribute 

polynomial a-factor correction matrices to the microanalysis community from our best Monte 

Carlo calculations of x-ray depth distributions for systematic binary element compositions at 

specific high specimen tilt angles. We plan to continue to test and refine these corrections and 

our analysis and quality assurance procedures by standards testing with our field emission 

scanning Auger and scanning electron microscopes. We plan to publish these methods once 

they have been refined and demonstrated to be robust. 

Accurate, Traceable Measurement of Samples of Low Electrolytic Conductivity 

K. W. Pratt and R. Shreiner (839) 

CSTL Program Area: Technologies for Future Measurements and Standards

This work was designed to obtain traceable measurements of samples with values of low 

electrolytic conductivity (κ) for κ values 

approaching that for pure water. Traceability 

for conductimetric measurements rests on the 

calibration of the given cell, i.e., the 

determination of its cell constant, Kcell. The 

original plan was to verify two independent 

methods for determining Kcell: (a) via a 

separate capacitance measurement in the 

same cell, and (b) via point-of-use generation 

of a chain of CO2-free transfer standards for 

electrolytic conductivity. The actual course 

taken is outlined below. 

CSTL researchers work to provide 

traceability for measurements of samples 

with values of low electrolytic 

conductivity (κ) for κ values approaching 

that for pure water. For these 

measurements, traceability rests on the 

calibration of the given cell, i.e., the 

determination of its cell constant, Kcell. 
167

The first hurdle, production of a temperature-controlled, CO2-free source of high-purity water, was 

overcome using an innovative coaxial coil. High-purity, CO2-free water is fed through PTFE 

tubing inserted inside metal tubing and through the annulus between these two tubes. The 

outside metal jacket serves as a CO2 barrier over the 10 m length of the coil. The PTFE tubing 

serves as a second barrier to exclude diffusion of ionic contamination (e.g., corrosion products 

from the metal tubing) into the cell feed water. At the downstream end of this coil, the Teflon 

tubing outlet feeds the conductivity cell. The annulus water goes to waste.

Using this approach and a flow-through conductivity cell, values for pure water were obtained that 

agreed to within 3% of literature values. Further measurements were deferred, pending 

construction of a working cell with Kcell optimized for pure water (κ ≈ 0.055 μS cm-1). It was 

further decided to construct an absolute cell, in which Kcell may be determined directly from 

physical measurements alone. Such a cell requires no calibration and yields independent 

verification of the two calibration methods noted above. 

The approach decided on was based on a historical design for an absolute capacitor [1] using 

guard cylinders to eliminate end effects (fringing). The outer electrode, with inside diameter di, is 

an unsegmented cylinder. The inner electrode, with outer diameter do, is 

segmented into three sections with narrow gaps perpendicular to the axis of 

the cell. The middle section, of length l, is electrically isolated from the two 

end (guard) sections. An AC potential is applied to the outer cylinder. The 

end sections of the inner electrode are grounded. The middle section is 

held at virtual ground. The current flowing through the middle section is 

measured to obtain the resistance, R. Requirements for inert electrodes, cost, total mass, and 

dimensional stability of the cell led to Ti as the material of construction. Measurements using this 

cell are expected in FY 2003. 

In developing the model for the absolute cell, it was essential to estimate the effect of the 

attainable dimensional tolerances on the overall uncertainty of the measurement of κ for pure 

water. To this end, a cylindrical capacitor was constructed in which the offset from coaxial 

geometry ("centering") could be accurately varied, to obtain the resulting effect on Kcell. Using a 

capacitor for such studies (in place of a conductivity cell) avoided practical issues associated with 

the use of liquid electrolytes. The results agreed well with new theoretical calculations. They also 

exposed shortcomings in the uncertainty 

analysis of the landmark classical study of 

an absolute coaxial capacitor, performed 

from 1904 to 1906 to verify Maxwell's 

Laws experimentally. 

Next Steps: A final milestone of this 

research was the realization that the 

dualism between the electrostatic field 

equations in a given capacitor and the 

corresponding equations governing an 

electrolytic conductivity cell with identical 

geometry applies to all geometries, 

provided that any nonideal field 

distribution (fringing) is the same in both applications. This principle, first discussed by 

Kohlrausch in 1906 but rarely exploited, subsequently, led to a fruitful series of discussions with 

Mike Moldover (CSTL’s Process Measurements Division) and may lead to future tests of his 

cross-capacitor geometries in a novel form of absolute electrolytic conductivity cell. 

This experiment had a spin-off result for pH SRM 

work: it demonstrated that alkaline pH buffers 

could be prepared using water taken directly 

from a point-of-use deionizing system without 

introducing a bias from CO2. SRM certificates for 

these buffers have recommended preparing the 

SRMs using source water that is boiled to expel 

dissolved CO2. This recommendation dates from 

the time when distilled (not deionized) water was 

used in laboratories. Since distilled water is CO2-

saturated, it must be boiled to expel the CO2

before use, whereas deionized water collected at 

a point-of-use system is CO2-free if adequately 

shielded from contact with the atmosphere. 

l R

d

d

1

2

ln

i

o

= •

π

κ
168

Nanowire Chemical Sensors Fabricated by Dip-Pen Nanolithography 

C.Taylor, M.J. Tarlov, and S. Semancik (836) 

CSTL Program Area: Technologies for Future Measurements and Standards

Dip-Pen Nanolithography DPN is a promising new technology, however, the selection of 

precursors and solvents is still a trial and error process and the present study was no exception. 

DPN of copper was first attempted because it was 

thought that copper lines would have a high enough 

conductivity to measure easily and would also be 

reasonably inert to atmostpheric oxidation. The 

strategy to form copper lines was to first write features 

of a copper salt and then reduce the copper salt in a 

hydrogen atmosphere to produce metallic copper. A 

CuCl2/n-propanol “ink” was found to possess several of 

the necessary solution properties to support DPN. 

First, the viscosity range was found to enable the writing of reasonably continuous lines. More 

viscous solutions were found to lead to splotchy, discontinuous lines, while less viscous solutions 

were found to yield exaggerated feature widths. Second, the n-propanol solution seemed to wet 

the surface of the cantilever well. Other solvents, such as water, did not wet the cantilever well, 

and the solution mixture did not adhere to the tip. Third, n-propanol acted as a good solvent 

allowing a high dissolved Cu(II) concentration (0.01mol solid/ 10 mL solvent mixture) and no 

flocculation was observed. A high CuCl2 concentration was required to write appreciable features. 

Later trials showed some promising preliminary results using Gum Arabic, but more studies are 

required to verify these results. 

The DPN experiments were performed in collaboration with Dr. Paul Sheehan of the Surface 

Chemistry Branch of the Naval Research Laboratory (NRL). NRL has Atomic Force Microscopy 

(AFM) capabilities that are ideally suited for the proposed DPN studies. The cantilevers were 

prepared by allowing them to soak in the solution for ~5 minutes. They were removed from the 

solution and blown dry using an air gun outfitted with a 

particle filter. The cantilever was then placed in the 

scanner head. The writing instrument was a 

Nanoscope 2 AFM. A rinsed and dried ≈1 cm × 1 cm 

piece of oxidized silicon wafer was used as the 

substrate for the DPN experiments. The cantilever was 

then scanned across the surface in a square-shaped 

pattern using a visual basic routine written at NRL. 

Following DPN writing, the samples were transferred to 

another AFM (ThermoMicroscopes model CP) for 

imaging. The use of a separate AFM for imaging the 

DPN features was advantageous because potential 

imaging artifacts from carryover of the CuCl2/npropanol “ink” during the imaging could be ruled out. 

Both DPN and imaging were performed in air. 

A typical AFM image of a DPN pattern performed with 

CuCl2/n-propanol “ink” is displayed in the figure. The 

cantilever was scanned linearly across the surface in a 

square-shaped pattern approximately 5.5 μm on a side 

at a scan rate of 1 Hz. The scale-bar in the image is 1 μm. The light, line-like features are 

approximately 100 nm or less in width and 5 nm or less in height. The longest continuous line is 

approximately 2 μm. We presume that these features are hydrated CuCl2 created by the DPN 

process. 

DPN of CuCl2 on a native 

oxide silicon surface. 

CSTL researchers aimed to 

fabricate metallic nanowires and 

investigate the conductometric 

gas sensing behavior of the 

nanowires using Dip-Pen 

Nanolithography (DPN), a new 

nanolithography method. 
169

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 2002, the following were formally recognized for 

their achievements. 

John Butler (831) was awarded the Presidential Early Career 

Award for Scientists and Engineers for advances in the fields of 

forensics and human identification. He also received the U.S. 

Department of Commerce Silver Medal for his contributions to 

the field of rapid and accurate DNA typing, particularly in support of 

human identification efforts in the wake of the terrorist attacks on 

the World Trade Center and the Pentagon. 

Lloyd Currie, CSTL Scientist Emeritus, was invited to become a Fellow of 

the International Union of Pure and Applied Chemistry (IUPAC). He 

was also awarded the Ioannes Marcus Marci Medal, which is the highest 

award of the Czech Spectroscopic Society of the Czech Academy of 

Sciences. 

David Duewer (839) received the U.S. Department of Commerce Bronze 

Medal for his innovative use of visualization techniques to summarize and 

communicate complex statistical information. 

Gary Gilliland (831) was awarded the U.S. Department of Commerce 

Silver Medal for establishing the Protein Data Bank as a unique 

international repository for structural data on biological macromolecules. 

William (Mickey) Haynes (838) was awarded the U.S. Department of 

Commerce Silver Medal for his leadership of the Physical and Chemical 

Properties Division of CSTL. 

Aaron Johnson (836) was recognized as a “Modern Day Technology 

Leader” for the 2002 Black Engineer of the Year Award. He was one of 

50 young engineers to receive a Certificate of Merit.
170

William Koch, Deputy Director of CSTL, received the U.S. Department of 

Commerce Silver Medal for establishing CSTL as a world-class laboratory 

within NIST. 

Chris Michaels, Lee Richter, and Stephan Stranick

(837) received the Samuel Wesley Stratton Award for 

scientific advances in the areas of visible near-field 

scanning optical microscopy (NSOM) metrology, Raman 

NSOM, and infrared (IR) NSOM. 

Archie Miiller (836) and his coworkers G. Cignolo (IMGC-CNR, Italy), M.P. 

Fitzgerald (MSL-NZ), and M.P. Perkin (NPL-UK), received the 2002 CSTL 

Technical Achievement Award for their paper “Final Report on Key 

Comparison CCM.P-K5 in Differential Pressure from 1 Pa to 1000 Pa” 

published in Metrologia 2002, 39, Tech. Suppl. 07002. 

Laurell Phillips (838) was selected as the first recipient of the CSTL 

Secretarial Achievement Award for his outstanding skills and providing 

extraordinary service to the staff and programs of CSTL. 

Ray Radebaugh (838) received the Federal Laboratory Consortium (FLC) 

Technology Transfer Award for his activities in collaborating with a small medical 

device company to develop cryogenic catheters that can freeze spots on the 

heart that cause heart arrhythmia. 

Stephan Stranick (837) received the U.S. Department of Commerce Bronze 

Medal for his research on developing near-field scanning optical microscopies 

(NSOM) as a nanometer-scale chemical probe of catalytic materials. 

Gregory Strouse (836) received the U.S. Department of Commerce Bronze 

Medal for his leadership in the dissemination of temperature measurements and 

standards and for establishing levels of comparability between U.S. national 

measurement standards and those of U.S. trading partners. 
171

Gloria Thomas (836) and Rose Hernandez (837) attended the 

52nd Annual Meeting of Nobel Laureates in Lindau, Germany. 

The meeting provides an opportunity for students and young 

researchers to meet with Nobel Laureates. 
172
The CSTL Divisions 

CSTL’s laboratory activities are primarily located at the NIST headquarters site in Gaithersburg, MD. We

also have research activities in NIST laboratories in Boulder, CO, and at the Center for Advanced Research 

in Biotechnology (CARB) in Rockville, MD. CARB is a collaborative effort of NIST, the University of 

Maryland, and Montgomery County, MD. 

Chemical Science and Technology Laboratory Physical and Chemical Properties Division

Dr. Hratch G. Semerjian, Director Dr. Gregory J. Rosasco, Chief 

(301) 975-8300 (301) 975-2483 

hratch.semerjian@nist.gov gregory.rosasco@nist.gov 

Dr. William F. Koch, Deputy Director 3⁄4 Basic reference data 

(301) 975-8301 3⁄4 Data for process and product design 

william.koch@nist.gov 3⁄4 Properties of energy-related fluids

3⁄4 Environmental fates of industrial chemicals 

3⁄4 Tools for chemical analysis 

Biotechnology Division 3⁄4 Fundamental studies of fluids 

Dr. Vince L. Vilker, Chief 3⁄4 Cryogenic technologies 

(301) 975-2629 3⁄4 Computational chemistry 

vincent.vilker@nist.gov 3⁄4 Basic measurements and standards

3⁄4 DNA chemistry, sequencing, and profiling 

3⁄4 Protein structure determination, properties, and Analytical Chemistry Division

modeling Dr. Willie E. May, Chief 

3⁄4 Biomaterials, biosensors, and bioelectronics (301) 975-3108 

3⁄4 Biocatalysis, bioprocessing measurements, and willie.may@nist.gov 

separations technologies 

Process Measurements Division

Dr. James R. Whetstone, Chief 

(301) 975-2600 

james.whetstone@nist.gov 

3⁄4 Research calibration services and provision of 

primary standards for temperature, pressure, 

vacuum, humidity, fluid flow, air speed, liquid 

density and volume, and gaseous leak-rate 

measurements 

3⁄4 Sensor research:

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3⁄4 Chemical measurements research and services in: 

Analytical sensing technologies 

Classical analytical methods 

Gas metrology 

Laboratory automation technology 

Nuclear analytical methods 

Organic analytical methods 

Spectrochemical measurement methods 

3⁄4 Tools for achieving international comparability of 

chemical measurements 

NIST Primary Reference Materials (SRMs) 

NIST Traceable Reference Materials 

(NTRMs) Micro-machined gas sensor arrays Interlaboratory proficiency testing programs Sensing applications of self-assembled 

monolayers International intercomparisons of 

measurement methods and standards 

Thin-film thermometry devices 3⁄4 Critical analytical data and specialty analyses Pressure, vacuum, and flow sensors Quantitative FTIR database for open path 

sensing applications 3⁄4 Measurements, models, and data for design and 

control of semiconductor and materials processing Aqueous solubility and Henry’s Law 

constants database for environmentally 

relevant compounds 

3⁄4 Engineering models and measurements for control

of thermal systems and industrial processes 

3⁄4 Multiphase chemical reaction engineering Analysis/Chemical characterization of high 

priority samples 

Surface and Microanalysis Science Division

Dr. Richard R. Cavanagh, Chief 

(301) 975-2368 

richard.cavanagh@nist.gov 

Visit the CSTL Website at: 

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

3⁄4 Nanoscale chemical characterization 

3⁄4 Particle characterization and standards 

3⁄4 Electronic and advanced materials 

characterization 

3⁄4 Chemical measurement process 

3⁄4 Surface and interface chemistry 

3⁄4 Advanced isotope metrology 

3⁄4 Molecular scale chemical characterization of 

organic and biomolecules 
 February 2003