NIST

PUBLICATIONS

NIST Special Publication 1012

AN APPROACH TO THE

METROLOGICALLY SOUND

TRACEABLE ASSESSMENT OF

THE CHEMICAL PURITY OF

ORGANIC REFERENCE

MATERIALS

NAT'L INST. OF STAND ft TECH

AlllOb flb2727

David L. Duewer

Reenie M. Parris

Edward White V

Willie E. May

Howard Elbaum

QCL

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- ulSI

National Institute of Standards and Technology • Technology Administration • U.S. Deportment of Commerce

NIST Special Publication 1012

AN APPROACH TO THE

METROLOGICALLY SOUND

TRACEABLE ASSESSMENT OF

THE CHEMICAL PURITY OF

ORGANIC REFERENCE

MATERIALS

David L. Duewer, Reenie M. Parris, Edward White V, Willie E. May

Analytical Chemistry Division

Chemical Science and Technology Laboratory

National Institute ofStandards and Technology

Gaithersburg, MD 20899

Howard Elbaum

AMSSB-RCB-RQ

U.S. Army Soldier and Biological Chemical Command

Aberdeen Proving Ground, MD 21010

September 2004

U.S. Department of Commerce

Donald L. Evans, Secretary

Technology Administration

Phillip J. Bond, Under Secretaryfor Technology

National Institute of Standards and Technology

Arden L. Bement, Jr., Director
Certain commercial entities, equipment, or materials may be identified in this document in order to describe an experimental procedure or concept adequately. Such

identification is not intended to imply recommendation or endorsement by the

National Institute of Standards and Technology, nor is it intended to imply that the

entities, materials, or equipment are necessarily the best available for the purpose.

National Institute of Standards and Technology Special Publication 1012

Natl. Inst. Stand. Technol. Spec. Publ. 1012, 53 pages (September 2004)

CODEN: NSPUE2
ABSTRACT

This document provides guidelines for the evaluation of the purity of organic chemicals,

usually high-purity chemicals, intended for use as reference materials and for the certification of

these materials such that assigned values can be considered as metrologically sound and

traceable to base units of mass and amount of substance.

The existing methodology for the analysis and certification of high-purity reference materials

is presented in sections on metrological concepts and nomenclature, procedures used for

preparation and certification of high-purity reference materials including analytical methods, and

the appropriate combining of different measurements. The consequences of decisions on

required purity, the nature of allowed impurities, traceability to other standards, quantitative

characterization, measurement uncertainties, and what information needs to be included in a

certificate are considered.

A general approach to the assessment of purity is proposed. The approach includes the

determination of what is required for a material to be fit for its intended use, the identification of

impurities, the determination of purity by direct or indirect means with a discussion of

appropriate analytical techniques, the combining of various analytical results, and the proper

presentation of available information. The certificates for a number of materials are evaluated

against this approach as case studies.

Four opportunities for research that would enhance the infrastructure available for methods

applicable to creating traceable high-purity organic standards are identified. These are: 1) the

creation of a limited set of certified materials for quantitative nuclear magnetic resonance, 2) the

generation and evaluation of data that would permit estimating uncertainties for measurements

based on relative signals from techniques such as gas chromatography and quantitative nuclear

magnetic resonance applied to the determination of impurities, 3) the improvement of the

prediction of chromatographic detector response factors through the use of quantitative structureproperty relationships, and 4) the development of a sensitive universal mass detector for liquid

chromatography with isotope ratio mass spectrometry and chemical reaction interface mass

spectrometry as promising candidates.

KEYWORDS

Chemical metrology, Chemical purity, Reference materials, Traceability

iii
iv
AN APPROACH TO THE METROLOGICALLY SOUND

TRACEABLE ASSESSMENT OF THE CHEMICAL

PURITY OF ORGANIC REFERENCE MATERIALS

TABLE OF CONTENTS

ABSTRACT iii KEYWORDS iii

TABLE OF CONTENTS v

GLOSSARY 1

1. INTRODUCTION 3

2. CONCEPTS AND NOMENCLATURE 4

2.1 Reference Materials (RMs) 4

2.1.1 Neat Materials 4

2.1.2 Solution Calibrants 4

2.1.3 Matrix RMs 4

2.2 Certified Reference Materials (CRMs) 4

2.3 Chemical Purity 4

2.4 Fitness for Purpose 5

2.5 Measurands 5

2.6 Calibration 6

2.7 Traceability 6

2.8 Measurement Uncertainty 6

2.9 Method Validation 7

2.10 Primary Method of Measurement 7

2.1 1 Components of Neat Chemical Materials 7

2. 1 1 . 1 Primary Component (PC) 7

2. 1 1 .2 Impurity Components (ICs) 7

2.12 Types of ICs 8

2.12.1 Critical Impurities 8

2.12.2 Suspected Impurities 8

2.12.3 Contaminants 8

3. BASIC PROCEDURE FOR DEVELOPMENT OF A NEAT-MATERIAL RM 9

3.1 Safety 9

3.2 Project Design 9

3.3 Suitability Analysis 9

3.4 Quantitative Characterization 10

3.5 Certificate Components for Neat-Material CRMs 11

v
4. METHODS OF CHEMICAL PURITY ANALYSIS 12

4.1 Types of Purity Analysis Methods 12

4.2 Direct Determination of the PC Amount 13

4.2.1 Gravimetry 13

4.2.2 Titrimetry 14

4.2.3 Internal Standard Quantitative Nuclear Magnetic Resonance (IS-qNMR) 14

4.2.4 Stable Isotope Ratio Mass Spectrometry 15

4.2.5 Cavity Ring-Down Spectroscopy (CRDS) 16

4.3 Direct Determination of the PC Mole or Mass Fraction 16

4.3.1 Area Percent Gas Chromatography (%GC) 16

4.3.2 Area Percent Liquid Chromatography (%LC) 17

4.3.3 Area Percent Quantitative Nuclear Magnetic Resonance (%qNMR) ...18

4.3.4 Molecular Spectroscopy 18

4.3.5 Elemental Analysis 19

4.4 Concurrent Determination of the IC Mole-Fraction 19

4.4.1 Thermal Methods 19

4.4.2 Phase Solubility Analysis (PSA) 20

4.5 Consecutive Determination of the IC Mole or Mass Fraction 21

5. COMBINING MEASUREMENTS , 22

5.1 Measurements of the Same Measurand Can Be Averaged 22

5.2 Measurements of Different Measurands Can Be Summed 23

5.3 Measurements of Not Quite the Same or Completely Different Measurands 24

5.4 Detection-Limited Quantitative Values 24

5.5 Dealing with Boundary Violations 25

6. AN APPROACH TO PURITY ASSESSMENT 27

7. CASE STUDIES 29

7.1 SRM 938 4-Nitrophenol 29

7.2 SRM 998 Angiotensin I (Human) 30

7.3 SRM 910 Sodium Pyruvate 31

7.4 LGC1 1 10 (pp'-Dichlorodiphenyl)dichloroethylene (pp'-DDE) 32

7.5 Arsenobetaine Master Material for BCR-626 33

7.6 BCR 289 2,4'-Dichlorobiphenyl (PCB-8) 34

7.7 NMD CRM 401 1 -a o-Xylene 35

7.8 Technical Profenofos 36

7.9 SRM 91 lb Cholesterol 37

8. OPPORTUNITIES 38

9. ACKNOWLEDGMENTS 39

10. REFERENCES 40

vi
GLOSSARY

AED Atomic Emission Detection

BCR Community Bureau of Reference, Brussels, Belgium

CRDS Cavity Ring-Down Spectroscopy

CRIMS Chemical Reaction Interface Mass Spectrometry

CRM Certified Reference Material, an RM with recognized pedigree

DSC Differential Scanning Calorimetry

ECD Electron Capture Detection

ELSD Evaporative Light Scattering Detection

FID Flame Ionization Detection

Fl Fluorescence detection

GC Gas Chromatography

IC Impurity Component

ICP Inductively Coupled Plasma ionization

ID Isotope Dilution

IRMS Isotope Ratio Mass Spectrometry

IS Internal Standard

Zi?95(quantity) Lower Bound of the 95% confidence interval on the specified quantity

LC Liquid Chromatography

LGC Laboratory of the Government Chemist, Teddington, England, UK

LoD Limit of Detection

NIST National Institute of Standards and Technology, Gaithersburg, MD, USA

NMIJ National Metrology Institute of Japan, Tsukuba, Ibaraki, Japan

mc mass (g) of any component of a material

m\c mass (g) of an IC

Mc relative molar mass (molecular weight, g/mol) of an IC

mPC mass (g) of the PC

MpC relative molar mass (molecular weight, g/mol) of the PC

MS Mass Spectrometry

NAA Neutron Activation Analysis

«c amount (mol) of any component of a material

"IC amount (mol) of an IC

NMR Nuclear Magnetic Resonance

"ndIC maximum likelihood estimate of the amount (mol) of a non-detected IC

"PC amount (mol) of the PC

OES Optical Emission Spectroscopy

PC Primary Component

PSA Phase Solubility Analysis

qNMR quantitative Nuclear Magnetic Resonance

QSPR Quantitative Structure-Property Relationship

RM Reference Material

SI Systeme International d'unites

SRM® Standard Reference Material, a CRM pedigreed by NIST

w(quantity) standard uncertainty (standard deviation) on the specified quantity

£/95(quantity) 95% expanded uncertainty on the specified quantity

c/B95 (quantity)Upper Bound to the 95% confidence interval on the specified quantity

1
UV/Vis UltraViolet-Visible absorbance detection

wc mass of substance fraction (g/g) of any component of the material

w\c mass of substance fraction (g/g) of an IC

wpc mass of substance fraction (g/g) of the PC

xc amount-of-substance fraction (mol/mol) of any component of the material

xic amount-of-substance fraction (mol/mol) of an IC

jcpc amount-of-substance fraction (mol/mol) of the PC

Y a specific measurand

Yj thej expected value of Y

%GC GC analysis based upon the ratio of peak areas %LC LC analysis based upon the ratio of peak areas %qNMR qNMR analysis based upon the ratio of signal areas

{Yj, U9s(Yj)} a nominally valid measurement result {expected value, 95% uncertainty} of Y

ZLoD sum of LOD for all relevant ICs

Zwic total mass (g) of all the ICs

L«ic total amount (mol) of all the ICs

Z«ndic maximum likelihood estimate of the total amount (mol) of all non-detected ICs

Zwic total mass of substance fraction (g/g) of all the ICs

Z*ic total amount-of-substance fraction (mol/mol) of all the ICs

2
1. INTRODUCTION

This report surveys the analytical techniques that have been used in the assessment of the

purity of organic chemicals. It proposes a general approach for purity characterization that

fulfills the requirements of, and should be recognized as, sound chemical metrology. When

conscientiously applied to the purity-characterization of a reference material (RM), this approach

traceably links the certified purity value of the RM to the base Systeme International d"unites

(SI) units of mass (kilogram) and amount of substance (mole) [1]. In combination with other

aspects of sound metrology, this linkage enables meaningful comparison of measurements made

in different laboratories and at different times.

Establishing the traceability of statements of chemical purity to SI units in principle requires

complete knowledge of the composition of the material analyzed. However, complete

knowledge of the chemical composition of any real substance is an impractical if not

unrealizable goal. Traceability of chemical measurements to the SI is therefore practically

realizable through substances appropriately characterized with respect to their chemical structure

(qualitative analysis) and composition (quantitative analysis) [2, 3]. These substances are usually high-purity (neat) materials.

Despite the central role of neat materials in establishing traceability, there is remarkably little

literature guidance on how to evaluate such materials for chemical purity - particularly for

structurally complex organic molecules. Formal requirements for assessing measurement quality

through method validation, uncertainly evaluation, and traceability demonstration are quite

recent, dating from the early 1990s [4]. However, the most complete review of analytical

methods useful for evaluating organic purity reflects the analytical capabilities of the late 1 960s

[5]-

Section 2 of this document introduces relevant metrological concepts and nomenclature.

Section 3 reviews the general RM development process, with an emphasis on how the evaluation

of neat materials differs from the evaluation of other chemical RMs. Section 4 presents and

evaluates the analytical methods that have been used or proposed for traceable analysis of neatmaterial RMs. Section 5 suggests ways of combining various types of measurement results.

Section 6 outlines an approach to purity assessment using analytical information from multiple

methods to achieve a metrologically sound traceable assessment of material purity. Section 7

discusses the assessment of several different neat-material RMs done in the past relative to the

approach proposed in Section 6. Section 8 presents four opportunities for enhancing the

analytical infrastructure for the traceable assessment of chemical purity.

3
2. CONCEPTS AND NOMENCLATURE

The following concepts and terms are essential or useful for discussing purity assessment

within the context of chemical metrology.

2.1 Reference Materials (RMs)

An RM is a "material or substance one or more of whose property values are sufficiently

homogeneous and well established to be used for the calibration of an apparatus, the assessment

of a measurement method, or for assigning values to materials" [6].

There are three basic types of RM used in chemical analysis:

2.1.1 Neat Materials

A neat material RM is a nominally single-entity material. These RMs are typically used to

prepare solution RMs and value assign matrix RMs.

2.1.2 Solution Calibrants

A solution calibrant is an RM prepared as a mixture of a diluent with one or more materials

of established chemical composition. These RMs may be intended for use as supplied and/or

following further dilution by the user.

2.1.3 Matrix RMs

A matrix RM is material of composition similar to that of "real samples" that contains a

characterized amount of one or more chemical entities. Matrix RMs are typically homogenized

natural materials with the entities of measurement interest present at endogenous levels,

augmented endogenous levels, or as added material.

2.2 Certified Reference Materials (CRMs)

A CRM is a "reference material, accompanied by a certificate, one or more of whose

property values are certified by a procedure which establishes its traceability to an accurate

realization of the unit in which the property values are expressed, and for which each certified

value is accompanied by an uncertainty at a stated level of confidence" [6].

2.3 Chemical Purity

While there is no formal metrological definition of either purity or impurity [7], the relevant

common usages are "the quality of being not mixed with anything else" and "the quality or condition of containing some extraneous or foreign admixture, especially of an inferior or baser

kind" [8]. More specifically: "A sample is sufficiently pure when its properties which are to be

investigated or used are representative of those of the main component within certain proscribed

limits of errors. In other words: a sample is sufficiently pure when the amount of each of the

impurities which may interfere with the specific purpose for which the sample is required is so low that their combined effect is negligible within the desired limits of accuracy" [9].

4
Thus, chemical purity is defined by the amount of desired material in a sample relative to the

total amount of all materials. The first step of purity determination is confirmation of the

chemical identity of the material. The degree of chemical purity that is acceptable is contingent

upon the needs of the intended use(s) of the material - i.e., its fitness for purpose. Materials are inadequately pure for use as RMs and CRMs when the impurities significantly influence the

chemical or physical properties of interest.

2.4 Fitness for Purpose

The fitness for purpose of chemical measurements is formally defined as the "degree to

which data produced by a measurement process enable a user to make technically and

administratively correct decisions for a stated purpose" [10]. A key element in the concept is for

the "interested parties to define in advance the acceptable degree of measurement uncertainty

and desired degree of identification confidence" [11].

In addition to being the criterion for assessing when any aspect of the measurement effort is

adequately complete, fitness-for-purpose considerations are central to the prospective design of a measurement study [12, 13]. The better defined the purpose, the more realistic the forecast of

analytical effort required to achieve fitness. An unrealistic, unclear, or overly broad purpose

may result in unnecessary costs, delay, or failure of a measurement study.

2.5 Measurands

A measurand is a "particular quantity subject to measurement" [6]. Within the context of

organic purity determinations, the critical measurands are the amounts of particular substances in a given sample.

Considerable effort may be required to define adequately the set of discrete chemical entities

that constitute a given measurand. Some of the definitional issues include:

• Isotopomers: Molecular entities differing only in isotopic composition. Unnatural

isotope abundances can affect the results of measurements performed using a wide

variety of analytical techniques. Unexpected isotopic abundances may arise from the

use of unusual source materials or be induced by manufacturing processes [14].

• Stereoisomers: Molecular entities having the same number and type of atoms and the

same bonds among the atoms but differing in the relative three-dimensional

orientation of various substructures. While some types of stereoisomers have

essentially identical chemical reactivities, all types of stereoisomers may have vastly

different biological activities. Nominally identical compounds prepared synthetically

and from biological sources may represent quite different sets of molecular entities.

• Rotamers and tautomers: Entities that interconvert with one another under typical

laboratory conditions but that differ in their relative two-dimensional orientation due

to restricted rotation or the nature of their bonds. While the discrete compounds may

have quite different properties, they often exist as equilibrium mixtures and are

difficult to isolate.

5
Fitness-for-purpose considerations dictate the descriptive detail required for appropriate

specification of amount-of-substance measurands.

2.6 Calibration

Calibration is a "set of operations that establish, under specific conditions, the relationship

between values of quantities indicated by a measuring instrument or measuring system, or values

represented by a material measure or a reference material, and the corresponding values realized

by standards" [6]. "It is the calibration process that transfers a reference value, usually an

International System (SI) unit, to the artifact or instrument under calibration and hence

establishes the 'unbroken chain of comparisons' required for traceability." [15]

2.7 Traceability

Traceability is the "property of the result of a measurement or the value of a standard

whereby it can be related to stated references, usually national or international standards, through

an unbroken chain of comparisons all having stated uncertainties" [6]. "[F]or consistent and

useful measurement results, it is important both that a chain of comparisons to agreed reference

standards, and the uncertainties associated with these standards, are established.'" [16]

Traceability is thus a way of ensuring that measurements made at different times, by different

analysts, or with different methods can be confidently compared.

Given a suitable RM for a particular chemical measurand, a traceable determination of the

amount of that measurand in a given test sample is straight forward - if exacting - analytical

chemistry. While requiring rigorously validated fit-for-purpose analytical methods and an

appropriate evaluation of the total uncertainty of the measurement, calibration to the RM

traceably links the measurement to the RM [15]. If the RM is a CRM (which, by definition, is

linked to a higher-order metrological standard), then the measurement is linked through the

CRM to that higher-order standard [2].

In the absence of an appropriate RM for a particular measurand, establishing traceability

requires linking the measurement either to some appropriate chemical comparator or to physical

property(ies) or physical principle(s) for which reference standard(s) are available [17, 18].

While typically requiring greater resources than direct calibration, such linkages are required

whenever the measurand of interest is insufficiently stable, when establishing a CRM, and when

the measurement uncertainty must be as small as possible.

2.8 Measurement Uncertainty

Measurement uncertainty is formally defined as a "parameter, associated with the result of a measurement, that characterizes the dispersion of the values that could reasonably be attributed

to the measurand. ... It is understood that the result of the measurement is the best estimate of

the value of the measurand, and that all components of uncertainty, including those arising from

systematic effects, such as components associated with corrections and reference standards,

contribute to the dispersion." [19]

6
2.9 Method Validation

Method validation is formally defined as "confirmation by examination and the provision of

objective evidence that the particular requirements for a specific intended use are fulfilled" [20].

The primary purpose of analytical validation is to "clearly define the application area and the

total reliability" of the methods [21].

2.10 Primary Method of Measurement

A primary method has been described as "a method having the highest metrological qualities,

whose operation can be completely described and understood, for which a complete uncertainty

statement can be written down in terms of SI units. A primary direct method measures the value

of an unknown without reference to a standard of the quantity. A primary ratio method measures

the value of a ratio of an unknown to a standard of the same quantity; its operation must be

completely described by a measurement equation." [22, 23] "First, it must be a method which is

specific for a defined substance and second, the values of all parameters, or corrections which

depend on other species or the matrix, must be known or calculable with appropriate

uncertainty." [17]

More recent commentaries recognize that there are no measurement methods or technologies

that operate at a primary level for all applications. Rather, there are a number of general

methods that have the potential of meeting the above requirements under specific circumstances

for selected measurands. The boundary conditions and scope of applicability for all such

methods must always be clearly stated and the method must be validated for the specific use

[24].

2.11 Components of Neat Chemical Materials

At some level, essentially all materials are mixtures. A neat material can be considered to be

a mixture of a primary component, PC, and a set of impurity components, ICs.

2.11.1 Primary Component (PC)

The PC is the chemical entity or aggregate of entities that is of primary interest. For neat

material RMs, the PC will be the dominant component of the mixture.

2.1 1.2 Impurity Components (ICs)

An IC is any chemical component of the mixture that is not the PC. There are two major

sources for ICs: chemical entities present in a material before purification and those that are introduced by purification efforts and material transfers. ICs in the original material that have

physico-chemical properties quite similar to those of the PC may be difficult to remove [9]. ICs

introduced into the material may have physico-chemical properties that are quite dissimilar to

those of the PC.

7
2.12 Types oflCs

While ICs can be classified in a number of ways, the following three potentially overlapping

categories are useful to following discussions.

2.12.1 Critical Impurities

Critical impurities are those whose presence at greater than some defined amount-ofsubstance fraction will render the material unfit for purpose.

2.12.2 Suspected Impurities

Suspected impurities are those that are at least plausibly in the material either from its known

history (especially preparation, purification, and storage) and its likely modes of

decomposition (especially thermal stability and reactivity with oxygen, water, and carbon

dioxide).

2.12.3 Contaminants

Contaminants are those substances that are not anticipated to be present in the material on

the basis of the material's known chemical properties or history. The presence of such ICs

suggests imperfect knowledge of the material's history and true properties.

8
3. BASIC PROCEDURE FOR DEVELOPMENT OF A

NEAT-MATERIAL RM

Development of a neat-material RM as a primary standard involves the same tasks as does

development of any other chemical RM. There is general agreement on the critical technical and

procedural requirements for valid RM certification [2, 3, 25 - 27]. The following sections review

the major considerations, with emphasis on those areas where the characterization of neatmaterial RMs may differ from practices applied to other types of chemical RMs.

3.1 Safety

This document is not intended in any way to cover or set policy or procedures for

environmental-, safety-, or health-related compliance or activities. However, it should be

recognized that neat organic materials must be handled with due care, in accordance with all

pertinent regulations and common sense. Determining the proper laboratory facilities, personal

protective devices, and environmental safeguards required for safe and responsible material

handling should be among the first considerations when starting any project.

3.2 Project Design

For a given problem, the components of the task must be clearly identified and assessed

against the available materials, methods, and resources. The PC and any critical ICs must be

explicitly identified and fit-for-purpose minimum (for the PC) or maximum (for ICs) amount-ofsubstance fractions established. Both "as pure as possible" and "zero tolerance" are unachievable within finite resources. Sufficient candidate material must be available to

accomplish the necessary certification studies and to provide an adequate number of RM units.

If fit-for-purpose candidate materials are not available, there must be sufficient resources

available to prepare or otherwise obtain fit-for-purpose material.

The physical and chemical properties of the PC must be compatible with the required

minimum shelf life for the standard under achievable storage conditions. Plausible qualitative

and quantitative methods of determination of the PC and critical IC measurands are required. If

specific methods are not available, there must be sufficient resources available to develop and

validate the needed techniques from known general approaches.

3.3 Suitability Analysis

Once one or more candidate materials have been identified, it is necessary to evaluate as

efficiently as practical their fitness for purpose. The material with the highest amount-ofsubstance fraction for the PC may not be the most fit; it may rather be the material that can be

most completely characterized - i.e., the material that can be assigned the smallest uncertainty

on the amount of PC or critical IC.

Confirmation of the identity of the PC is a central task of purity determination. All validated

techniques appropriate for the determination of the identity of the PC should be considered.

9
When the physical properties of the material are well-known and distinctive, evaluation of one or more characteristic property such as melting point may suffice. In the more general case, various

specialized forms of high-resolution chromatography, nuclear magnetic resonance (NMR), mass

spectrometry (MS), and molecular spectroscopy (ultraviolet, visible, infrared, and Raman) may

be required.

The presence/effective absence of critical ICs should be assayed and, if detected, the amount

of each material at least semi-quantitatively evaluated. All validated techniques appropriate to

this purpose should be considered. Typical techniques for organic ICs include many of the

"hyphenated" combinations of separation technologies and spectroscopic detection such as gas

chromatography with flame ionization (GC-FID) or MS detection (GC-MS) and liquid

chromatography (LC) with MS detection (LC-MS). Typical techniques for inorganic ICs

include X-ray fluorescence spectrometry, inductively coupled plasma ionization (ICP) in

combination with optical emission spectroscopy (ICP-OES) or MS (ICP-MS), neutron activation

analysis (NAA), ashing, and - for water - mass loss on drying or Karl Fischer titration.

Identification and quantitative determination may be accomplishable from the same assays.

Once suitable techniques have been established, a worst-case material heterogeneity survey

should be conducted. This study should compare several "as different as possible" sub-samples

of the material. This may include different containers, different strata (top, middle, bottom)

within a single container, and different particle sizes or crystal morphologies. If the material is

appreciably heterogeneous, the material should be purified and/or blended to establish adequate

uniformity. A minimum sample size for reliable analysis should be identified. This survey may

be accomplishable along with quantitative characterization.

3.4 Quantitative Characterization

The quantitative characterization of a neat material does differ from that of other RMs. First,

in nearly all cases any measurement of the PC must be accomplished via calibration to a

chemical comparator different from the PC or to some physical property or principle. Second,

the characterization may require many different types of measurements of different chemical

components and/or properties. This requires a flt-for-purpose evaluation of the identity of the

PC and the nature and amount of impurities in the material. Since "absence of evidence is not

evidence of absence" (Sir Martin J. Rees, UK Astronomer Royal), the realistic goal is to

assemble a chemically convincing body of evidence that the evaluation

• would have observed all critical and suspected ICs if they had been present at

significant levels relative to the material's intended purpose,

• included a sufficiently broad survey for contaminant ICs,

• included a sufficiently sensitive blunder-check comparison of direct and indirect

determinations to catch any major overlooked ICs or errors of analysis, and

• the results of all determinations have been appropriately combined (see Section 5).

As with any RM, the homogeneity of the material must be evaluated. As a general rule, the

homogeneity study should evaluate the ICs that are present in largest amount, are expected to be

most variable or innately susceptible to environmental contamination (such as water), and/or are

10
most critical to fitness for purpose. Comparison of multiple lots or sources of the material can

help to identify the most variable ICs.

The lifetime and expected shelf life of the material must also be evaluated. These may be

assessed from the material's physical and chemical properties and through accelerated aging

experiments. Aged samples should be evaluated for any qualitative and quantitative changes in

the ICs present.

3.5 Certificate Components for Neat-Material CRMs

Ultimately, an RJvl's utility is determined by its fitness for purpose for a particular

measurement and the degree of confidence the user of the RM has in the assigned value(s). In

addition to appropriately stating the amount and uncertainty for the PC and other formal

requirements [28], to enable a user to appropriately assess the utility of the CRM for a particular

purpose the certificate should also:

• state the identity and amounts of all ICs that were quantitatively determined,

• state the identity and limit of detection for other ICs for which the material was

explicitly evaluated,

• identify the analytical techniques used to assess the material, including those used to

determine identity and to survey for contaminants,

• describe the assumptions and methods used to assess uncertainties and to combine

individual results, and

• explicitly assert the traceability of the certified values.

11
4. METHODS OF CHEMICAL PURITY ANALYSIS

The following discussions summarize the major strengths and weaknesses of analytical

technologies for neat-material purity determination. The discussions are not intended to present

complete procedures. Many "analytical details" are quite specific to given technologies; the

provided references are intended as introductions to the relevant literature. However, some

aspects of the analytical process, particularly sample preparation and the selection of

representative blanks and controls, are essential for achieving accurate analytical measurements

with nearly any technology. Measurement artifacts can be introduced from incomplete

processing or introduction of impurity components of reagents and solvents, from contact with

container walls, and from environmental exposure. An introduction to these essential if generic

aspects of the analytical process is provided in Kolthoff s Treatise on Analytical Chemistry [29].

4.1 Types of Purity Analysis Methods

The amount of PC, «pc, plus the amount of all the ICs, Z«ic, by definition is equal to the total

amount of all substances in a sample. Likewise, the mass of PC, mpc, plus the mass of all the

ICs, Z>wic, is equal to the total mass of all substances in a sample. Given a well-characterized

PC, amount of substance and mass of substance are readily converted through the PC's relative

molar mass (molecular weight), Mpcm

pc =npc *Mpc . [la]

Similarly, the amount of substance and mass of substance of every individual IC are converted

through each compound's relative molar mass, M\c.

mlc =nlc xMlc . [lb]

As long as units are consistent, amounts of substance and mass of substance are meaningfully

expressed as fractions of the total sample. The amount-of-substance fraction of a component C

(either the PC or an IC), xq, is defined as

*c = [2]

npc

+

and the mass of substance fraction of a component C, wq, is defined as

TMc = £ • [3]

m

pc +2^ic,

The xc and wc will in general not be equal unless all components have the same Mc, however,

they do become closer in value as the Mcs become similar and the total amount of impurities

becomes small relative to the amount of the PC.

12
Note that the amount-of-substance fractions and the mass of substance fractions sum to unity

If all of the ICs present in the sample can be accounted for, there are four different approaches to

determining the purity of a neat material: direct estimation of «pc, direct estimation ofxpc or wpc,

concurrent estimation of 1 - 2jcic, and consecutive estimation of 1 - Z*ic or 1 - Zwic4.2 Direct Determination of the PC Amount

The following technologies estimate «pc without necessarily quantifying all of the ICs.

4.2.1 Gravimetry

Gravimetry is not a single method, but rather a collective term for many quite chemically

different techniques that have in common the determination of the «c of a (highly soluble)

measurand as a weighable mass of a (highly insoluble) reaction product [29, 30]. Classical

gravimetry is distinct from and should not be confused with gravimetric preparation (preparing

an RM by mixing weighed quantities of two or more well-characterized materials), gravimetric

titration (metering a titrant by mass rather than volume), or thermogravimetric analysis (mass

loss as a function of temperature).

As they rely on accurate weighing and material transfers, gravimetric techniques for organic

compounds require destruction of tens of milligrams to many grams of sample. Relative

standard uncertainties of less than 0.1% relative to the mass of the precipitate can be achieved.

The methods can be made valid for any «pc- As the «ic are not determined but Mpc is known,

wpc is the most valid form of fractional determination.

While the basic gravimetric principle depends upon very specific reactivities, relatively few

organic compounds can be gravimetrically determined without first transforming them to another

chemical entity. PCs that have specific functional moieties or heteroatoms may be transformable

into gravimetrically determinable entities, e.g., quantitative oxidation of a sulfur-containing

measurand into inorganic oxides (including SO3), dissolution into an appropriate solvent

followed by precipitation with excess barium chloride, and gravimetric determination of the

resulting barium sulfate. However, such conversions are typically not specific; in the example,

all sulfur-containing compounds originally in the material will be summed into the final sulfate

measurand.

The results of gravimetric measurements are nominally traceable through the mass

calibration of the weighing system and the use of transformations that provide a known, fixed

stoichiometry for the insoluble reaction product. However, few chemical transformations are

entirely specific or complete. In addition to requiring the identification and at least semiquantification of interferent ICs, corrections for losses in any preliminary chemical

transformations, incomplete precipitation, co-precipitation, volatile losses, filtration losses, etc. are required. In their purest forms, gravimetric methods are therefore of little practical utility for

the traceable determination of organic purity. However, these corrections and their uncertainties

may be amenable to determination using fit-for-purpose instrumental methods [31, 32].

[4]

13
4.2.2 Titrimetry

There are several chemically different classes of titrimetric methods and a vast number of

specific techniques within each class [29, 30, 33 - 35]. Coulometric titrations determine n?c via

reaction of the measurand (or some chemical transformation of the PC) with metered charge

transfer. Gravimetric titrations determine «PC via reaction with a chemical titrant metered by

mass. Volumetric titrations determine n?c via reaction with a chemical titrant metered by

volume.

As they rely on accurate metering of the reactant, titrimetric techniques require milligram to

gram quantities of sample. The analyses destroy the sample or render it unfit for further

characterization. Relative measurement uncertainties of as low as 0.01% can be achieved. The

methods can be made valid for any «pc. As the nic are not determined but MPc is known, wPC is

the most valid form of fractional determination.

Most organic PCs that contain electrochemically or chemically reactive functional groups

can be determined with some type of titration, either directly or after chemical transformation.

Titrimetric techniques are typically quite functional-group-specific but not compound-specific;

any IC that contains a moiety of similar reactivity to that of the PC will consume titrant.

The results of titrimetric measurements are nominally traceable through the calibration of the

metering system and, for volumetric and gravimetric methods, through the calibration

(standardization) of the titrant. As with gravimetry, these measurements may require correction

for non-ideal chemical transformations as well as for interferent ICs. Such corrections and their

uncertainties may be amenable to determination using fit-for-purpose instrumental methods [30].

4.2.3 Internal Standard Quantitative Nuclear Magnetic Resonance (IS-qNMR)

When immersed in a strong magnetic field a number of atomic nuclei interact with radiofrequency electromagnetic radiation in a manner that, to an excellent first approximation, does

not depend upon the chemical composition or bonding of the molecule that contains the nuclei.

The fundamental sameness of the transition frequency of each nuclear type is modified by

second- and third-order atomic interactions related to their bonding and three-dimensional

orientation. These small differences become better resolved and the transition intensity more

readily quantified as the magnetic field strength is increased.

The chemical structure-related differences in the transition frequencies make NMR one of

the central tools for molecular structure elucidation. Conversely, the almost-but-not-exactlyidentical transition frequencies enable comparison of the number of same-type nuclei in a given

chemical environment with the number in another chemical environment. While most

commonly used to estimate the relative numbers of nuclei in different chemical environments

within one type of molecule, inclusion of an appropriate internal standard (IS) within the sample

enables determination of the relative numbers of different types of molecules in the sample

[36 - 41]. The internal standard can be of quite different structure from the measurand as long as

it contains one or more nuclei of the relevant atomic type. The IS can be mixed with the

measurand or, to prevent reaction between the measurand and the IS, it can be isolated inside a

coaxial insert within the sample holder [41 - 43]. While potentially complicating the analysis, a

variety of solvents are available for use with solid or viscous samples [42].

14
IS-qNMR methods are valid for any wpc level. As the «ic are not determined but Mpc is

known, wpc is the most valid form of fractional determination.

The Table below lists commonly exploited NMR-active nuclei, their typical natural

abundances and relative sensitivities, and an indication of how frequently they have been used in

qNMR studies. Most organic materials contain one or more of these nuclei. Typically a sample

size of a few to several hundred milligrams is required. Isolation of the IS from the measurand

allows recovery of the measurand for other studies. Relative measurement uncertainties of 0.5%

and less are achievable [44].

NMR-Active Nuclei Used in qNMR Studies [45]

Nucleus

% Natural

Abundance

Sensitivity Relative to

Same Number of ! H

Atoms

Abundance x Relative

Sensitivity

Number

qNMR

Publications

lH 99.98 1 1 Many

,9F 100 0.833 0.8 Few

31p 100 0.066 0.07 Several

17o 0.037 0.029 0.001 Several

29

Si 4.70 0.008 0.0003 Few

13 C 1.11 0.016 0.0002 Many

15N 0.37 0.001 0.000004 None found

The results of single-pulse IS-qNMR measurements are traceable to the amount of substance

of the internal standard used. The quantitative utility of multiple-pulse methods using

decoupling or cross polarization techniques of any sort must be carefully evaluated on a case-bycase basis. Since the actual comparison is the number of nuclei in one chemical environment

relative to the number in a second environment, only one isolated signal each from the IS and the

material of interest is required for comparison. However, all of the relevant NMR transitions for

both materials should be characterized prior to selecting the particular transitions to be used in

the comparison.

4.2.4 Stable Isotope Ratio Mass Spectrometry

There are a number of MS techniques that can be used use to characterize quantitatively

components in a mixture, all using the ratio of signals from two or more stable isotopes of given

atomic types. Isotope dilution MS (IDMS) techniques exploit the change in ratios as a function

of the addition of known quantities of an isotopically enriched version of the same measurand.

While often sufficient in itself for the analysis of calibration solution and matrix RMs [46 - 48],

one-step IDMS has limited applicability for the determination of «pc for neat organics since it requires detailed characterization of the labeled material. Multiple-step IDMS may enable

"analyses for which reference materials with certified isotope ratios are not available" [49].

Relative uncertainties of a few 0.01% to 0.1% are achievable.

Relative uncertainties of a few 0.1% can also be achieved by measuring the ratios of reaction

products using isotope ratio MS (IRMS) or chemical reaction interface MS (CRIMS) where the

15
molecules of interest are converted online to small polyatomic molecules such as CO2, NO, and

SF6 [50, 51]. Used in combination with chromatographic separations, these methods can be

calibrated with suitably labeled comparators that are structurally different from the desired

measurand.

Results of measurements made using the isotope ratio MS techniques are traceable to or through the isotopically enriched comparator and, most importantly, to the complete

experimental realization of the assumptions of the relevant complete measurement equation.

4.2.5 Cavity Ring-Down Spectroscopy (CRDS)

CRDS is a relatively new optical spectroscopy that exploits the time-domain decay of light

trapped within an extraordinarily reflective resonance cavity to determine the product of the

number density of a specific gaseous absorber and its absorption cross-section. Once the cross- section is known, «pc can be deduced from measurement without direct use of a chemical

comparator.

While CRDS has been represented as a "primary method" for the analysis of trace gases [52,

53], the requirement for accurately determined absorption cross-section suggests that it has very

limited applicability for the analysis of neat materials. That is, materials suitable for adequately

determining the cross-section would be adequate for the calibration of other analysis techniques.

As currently developed, CRDS is suitable only for the determination of molecular entities that

have narrow absorption features for which single-mode laser sources are available that can be

tuned completely across the feature.

Results of CRDS measurements are traceable to the RM used to define the absorption crosssection.

4.3 Direct Determination of the PC Mole or Mass Fraction

If all components of a mixture could be completely resolved as a function of time (for

separation systems), frequency (for optical spectroscopic systems), mass (for mass spectroscopic

systems), or other physical principle and if each component could be detected with equal molar

or molar-mass sensitivity, then the ratio of the signal due to the PC relative to the sum of the

signals from all components would directly estimate xpc or wPc. The following technologies

approximate this direct estimation without necessarily quantifying all of the ICs.

4.3.1 Area Percent Gas Chromatography (%GC)

A well-designed and correctly executed determination using capillary column GC can

provide very complete separation of mixture components. IRMS, CRIMS, and atomic emission

detection (AED) quantitatively convert molecules to their constituent atoms or to a set of

characteristic small polyatomic molecules and thus have the potential to be highly sensitive

universal molar-mass detectors [50, 51, 54]. These techniques require specialized equipment and

expertise; their quantitative performance characteristics have not yet been thoroughly

characterized. Of the commonly used detection principles, flame ionization detection (FID)

comes closest to providing an adequately sensitive response that is proportional to the relative

molar mass [55].

16
FID is quite sensitive for any volatile or semi-volatile molecule that contains C-H linkages,

has a very large linear dynamic range, and is proportional - to a good first approximation - to

the carbon content of the molecule. When all ICs are known to be very similar to the PC (i.e.,

isomers and close homologues), GC-FID directly estimates wpc [56, 57]. Given a suitably

volatile material, capillary column GC analysis of a neat material requires very little sample.

Relative integration uncertainties of 0.5% can be achieved. Since the GC-FID (1) is insensitive

to water and non-volatile compounds and (2) provides a response to volatile compounds that is

only approximately constant even for isomers and close homologues, this approach is never

completely valid. Ongoing research into chemical class-specific response factors, linearity of

response factors, and associated detection uncertainties may enhance the utility of %GC-FID for

the assessment of neat-materials [58].

Results of %GC wPC measurements are traceable through the validity of the assumptions:

• the wpc is very high,

• the chromatographic separation achieves complete separation of all ICs from the PC,

• all of the ICs elute and are detected, and

• the detector response factors are very similar for the PC and all ICs.

Use of capillary columns, temperature programming, and several different column stationary

phases can help validate the suitability of the separation. GC-electron capture detection (GCECD) provides excellent sensitivity for halogenated compounds. GC-MS with library search

capability can help confirm the similarity of ICs. GC-IRMS and GC-CRIMS may enable

calculation of GC-FID response factors even if molecular structures cannot be uniquely

identified (see Section 4.5).

4.3.2 Area Percent Liquid Chromatography (%LC)

While LC systems typically provide somewhat lower resolution than can be achieved with

capillary GC, they enable use of a much greater diversity of stationary and mobile phases.

Except for very volatile materials, high-performance LC systems may thus enable more complete

separation of components than can be achieved with GC alone. Typical LC separations are performed at lower temperatures than GC and are thus less prone to thermal degradation of the

sample material. However, no LC detection system is available that is suitably sensitive and that

responds about equally to all organic material.

The widely used ultraviolet-visible absorbance (UV/Vis) and fluorescence (Fl) detectors

provide excellent sensitivity for many organic moieties; however, many compounds do not

contain UV/Vis- or Fl-active substructures. Further, LC-UV7Vis and LC-F1 response intensities are sensitive to small changes in molecular structure; the common analytical practice of using

single-wavelength UV/Vis detection is inappropriate for characterization of unidentified ICs.

Evaporative light scattering detection (ELSD) has been claimed as a step towards a universal

LC detection principle [59]. An ELSD detector consists of a nebulizer to convert the eluent to an

aerosol, a drift tube to vaporize the liquid-phase solvent, and a light-scattering cell where the

detection of any particles takes place. However, LC-ELSD is much less sensitive than GC-FID

and has a smaller linear dynamic range. The scattering signal is a complex and not fully

understood function of the particle size and how each compound absorbs, refracts, reflects,

17
Rayleigh scatters, and Mie scatters the incident light [59]. The net response factors do not yet

appear to be quantitatively predictable. At present, LC-ELSD appears to be most useful for

semi-quantitative confirmation studies.

While unproven for purity determination, LC-IRMS and LC-CRIMS have the potential for

estimating the relative mass as well as at least partial molecular formulas for all ICs [50, 60].

4.3.3 Area Percent Quantitative Nuclear Magnetic Resonance (%qNMR)

%qNMR compares the integrated area for all of the NMR nuclear transitions for a given

atomic type that are attributable to the PC to the sum of the area of all observed transitions of the

material. If at least one but not all of the PC transitions can be resolved from those of the ICs,

the area ratio can be established by suitably correcting the measured area by the ratio of total

nuclei to measured nuclei [42].

Unlike the separation-based GC-FID and LC-ELSD methods, all ICs that contain the

relevant isotope will contribute to the total integral. To a good first approximation, each relevant

atom in the material contributes equally to the total signal intensity. When the relevant isotope is

present in very different chemical environments, the relative response factor for each transition

frequency is a calculable function of the difference between the transition and the fundamental

excitation frequency [39]. If the ICs can be identified, the appropriate atom-to-molecule ratios

can be determined and corrected for (see Section 4.5). Relative integration uncertainties of 0.5%

can be achieved, although ICs present atxpc of less than «0.1% may not be fully accounted for.

1 13 Comparison of results for different atomic types, particularly H- and C-NMR, can assist

validation. The results of %qNMR xpc measurements are traceable through the validity of the

assumptions:

• the xpc is very high,

• all signals have been completely integrated, and

• the atom-to-molecule ratios have been correctly determined.

4.3.4 Molecular Spectroscopy

If the molecular spectrum (typically in the ultraviolet, visible, infrared, or near infrared

spectral regions) of a material is known then jcpc may be determinable by comparison of an

observed spectrum to the known spectrum. If the spectrum of the PC contains one well-defined

unique feature then xpc can be calculated as the ratio between the observed and the known signal

for the feature. This is known as zeroth-order calibration; it is appropriate only when none of the

potential ICs have any spectral signal in the region of interest. When no single feature provides

adequately selective information, a variety of multivariate mathematical techniques may enable

estimation of x?c via comparison of multiple features. This is known as first-order calibration; it

is appropriate when the spectra of all the potential ICs are known. When two or more molecular

spectroscopies are used to simultaneously generate a matrix of information, as in molecular

excitation/emission spectrofluorometery, then recent developments in multivariate chemometrics

may enable estimation ofxpc in the presence of unknown ICs. This is known as second-order

calibration or N-way analysis [61 - 63].

18
Since zeroth- and first-order calibrations require the absence of, or at least knowledge of the

spectral characteristics of, all interferences, they cannot be used to directly estimate xpc -

although they may be useful for consecutive determinations as described in Section 4.5. Only

second-order calibration systems are in principle appropriate for direct estimation of PC purity.

The precision that can be achieved is a predictable function of the spectral characteristics of the

PC, the particular spectroscopic system, and the mathematics used [64]. Relative uncertainties

of a few 0.1% or less have been achieved. Second-order spectroscopic calibrations are traceable

to the RM used to define the reference spectrum.

4.3.5 Elemental Analysis

There are many techniques that can be used to assay the mole fraction elemental composition

of most organic materials [65]. Relative uncertainties of a few 0.1% have been claimed for the

determination of C, H, N, O, P, S, As, CI, Br, I, and Se. Such assay is very useful as a blunder

check - deviation of the observed composition from that expected strongly suggests the presence

of contaminant ICs of quite different composition from the PC. However, elemental assay is of

no utility for revealing the presence of ICs having the same or very similar molecular formula as

the PC.

4.4 Concurrent Determination of the IC Mole-Fraction

The following colligative technologies have been claimed to estimate 1 - Xjcic without

identifying any of the ICs.

4.4.1 Thermal Methods

Given a material that is crystalline at an experimentally accessible temperature, there are numerous methods that directly estimate Jjcic by measuring various phase-change related

phenomena as a function of temperature [66 - 69]. The more commonly used of these methods

are: freezing- and melting-point depression, differential scanning calorimetry (DSC), and

adiabatic calorimetry. These methods determine the degree of deviation from the behavior

expected of an ideal 100% pure sample of the material caused by the presence of impurities that are more soluble in the material's liquid phase than in its solid phase. They are applicable when

the "pure" material is stable at its melting point and all impurities are soluble in the liquid phase.

Freezing-point and melting-point techniques monitor the fraction of material transformed

from one phase to the other as a function of temperature. DSC techniques monitor the

relationship between the relative heat flow and the temperature of a sample in comparison to an

inert reference. Adiabatic calorimetric methods monitor the absolute heat capacity of a material

as a function of sample temperature. Other related techniques monitor volume and dielectric

changes in a material as it melts or freezes. These methods generally require 0.001 g to 0.1 g

amounts of sample. The quantitative physical relationships upon which these techniques are based become more rigorously applicable as Zxic goes to zero. Precisions of a few 0. 1% or

better can be achieved at xpc of «0.99 or greater. All of the methods become progressively less

accurate with declining xpc; the limit of quantitative utility is «0.95.

19
The results for all of the thermal methods assert traceability through the validity of their

assumptions:

• the xpc is very high,

• the experiments realized the required thermodynamic conditions,

• there are no insoluble ICs, and

• there are no ICs that form solid solutions.

The differential techniques also require one or more calibrator RMs. DSC has been claimed as a "primary method" for purity [17]; however, in practice DSC often involves empirical corrections

that, while of great practical utility, decrease the method's claim to produce traceable results

[70]. Of all the current methods, adiabatic calorimetry can best experimentally satisfy its

theory's assumptions; it is also the most time and resource intensive [66].

Unfortunately, all colligative techniques are insensitive to ICs that form solid solutions with

the PC; i.e., entities that are soluble in both the solid and liquid phases of the PC. Unless the ICs

are identified and determined not to be likely to co-crystallize with the PC, the relationship

between the character of the phase transition and absolute material purity cannot be adequately

established.

4.4.2 Phase Solubility Analysis (PSA)

Given a material that is. sparingly soluble in some well-defined solvent system and that is

crystalline at a temperature that can be maintained quite constant for several weeks, PSA can be

used to both estimate Yjcic and to concentrate the individual ICs for further characterization.

PSA monitors the composition of the liquid phase in a series of sealed vials containing

increasing amounts of material in fixed amounts of solvent, kept at constant temperature and

pressure, as a function of system composition (the amount of the material per unit mass of the

material plus the solvent) [71, 72].

While it is non-destructive, PSA requires large amounts (1 g to 100 g) of sample and several

weeks of elapsed time to perform. Due to its dependence on the use of discrete systems, the

upper limit to the xpc that can be resolved is «0.999. ICs that are more soluble than the PC in the

chosen solvent tend to be concentrated into the solvent and thus may be more easily identified in

these solutions than they were in the original material [73]. The solvent systems must not

chemically react with the PC or any of the ICs.

Like the thermal colligative methods, the results of PSA measurements are traceable through

the validity of its assumptions. When care is used to ensure that equilibrium conditions are obtained for all of the discrete systems, the necessary conditions can rather simply be realized for

many materials. However, again like the thermal colligative methods, PSA is insensitive to ICs

that form solid solutions. Use of two or more solvent systems can help reveal such problems

[74].

20
4.5 Consecutive Determination of the IC Mole or Mass Fraction

Any and all validated analytical methods may prove useful for the identification and

quantitation of ICs. Some composite ICs such as "insoluble particulate matter," "volatiles," or "ash" may be determinable using gravimetric measurements. More generally, specific ICs need

to be individually evaluated using appropriate instrumental methods. However, it is unlikely that

suitable RMs for all (or most - or any) of the ICs will be available for calibrating any of the

methods. How then can these methods be useful in a purity analysis of a neat material?

The "trick" is that the ICs are (rather, should be) present in the material in amounts several

orders of magnitude less than the PC. Large relative uncertainties in the determination of small

amounts of material may give adequately small absolute uncertainties [31, 34].

Assume that some %area method, such as %GC-FID, can be expected to give results having

a relative standard uncertainty of 5%. If wpc = 0.99, the 95% expanded uncertainty on wpg

£/95(wpc), is equal to ±2 * 0.99 x 0.05 = ±0.1; this is unlikely to be adequate. However, if used

to analyze an equally "high-purity" calibrant material for some IC that is present at w\c = 0.01

then the t/95 (wiC) is equal to ±2 x 0.01 x 0.05 = ±0.001. In general, the lower the amount of the

IC present in the candidate RM (relative to the amount of the most abundant IC detected), the

less critical the expected uncertainty in the analysis of the IC.

Thus, given a sufficiently pure candidate RM so that the IC amounts are small, commercial

materials can probably be analyzed sufficiently well for use as fit-for-purpose IC calibrants. It is

likely that materials that are adequate to establish confidently the identity of the ICs will also be

adequate for their calibration.

When an IC has been identified but no commercial material is available within the time and

resource constraints of the project, it may be possible to calculate an appropriate response factor

for some methods. There are a number of software systems that enable the calculation of NMR

spectra from first principles. Several different quantitative structure-property relationship

(QSPR) studies have predicted GC response factors for FID, thermal conductivity, and

photoionization detection systems to within about 5% [75 - 77].

The result of a consecutive 1 - Z*ic or 1 - Zwic determination is traceable through the

validity of the assumptions:

• all ICs (or chemical class groups of ICs) have been recognized,

• all ICs have been appropriately quantified, and

• the results of the multiple analyses have been appropriately combined.

21
5. COMBINING MEASUREMENTS

When no single measurement proves fitness for purpose, chemical purity must be estimated

by assembling information from different sources. Combining disparate data into a

metrologically acceptable and chemically satisfying conclusion is an exercise in chemical

deduction. It is essential that appropriate mathematics be employed, but determining what

information can be combined is primarily a scientific - not a statistical - challenge, task, and

responsibility.

5.1 Measurements of the Same Measurand Can Be Averaged

When the identities of all the detected ICs are known and the amounts of each have been

individually determined, it is relatively simple to:

• identify with fit-for-purpose confidence any replicate measurements of the same

quantity,

• transform, if necessary, such replicate measures to have consistent units, and

• composite replicates to yield a single quantitative estimate - an expected value and

an associated 95% confidence interval about the expected value.

Given Nnominally valid estimates from different analytical methods for measurand Y, how

should they be combined to provide the most meaningful estimate of the true value of Y?

Let {Yj, U9s(Yj)} represent the set of Admeasurements, where Yj is the best estimate of the

value from methodj and U^siYj) is its fully evaluated 95% expanded uncertainty [19]. When the

YjS substantially coincide (i.e., when the results from different methods agree within their

assigned uncertainties) and the U^Yj) are approximately equal, an adequate estimate is provided

by the simple mean of the methods

N

Y=^Yj N [5]

;=1

and the approximate expanded standard uncertainty of the mean (combining the among-method

and the average within-method variances)

UQ5 (Y) = 2x

i

N i \o I I N

>

* ( N fll /V »\ 2

W=1 J w'=1

uQ5 (Yj)

N

[6]

The use of the value 2 as the coverage factor to convert combined standard uncertainties of fully

evaluated measurements into approximate 95% coverage expanded uncertainties is inexact but

quite conventional and accepted metrological practice [19]. If the Nmeasurements must be

regarded as random draws from a population of possible measurements (e.g. , results from an

interlaboratory study), a Student's t coverage factor for AM degrees of freedom may be more

appropriate.

22
When the YjS substantially coincide but the U^Yj) are very different, an inverse totaluncertainty weighted mean may be a better estimate than the simple mean [78, 79]. (Total

uncertainty includes all among-method as well as within-method uncertainty components.)

However, qualitative and semi-quantitative confirmatory results are valuable primarily to

demonstrate the lack of bias of the more quantitative measurements. While all available

information needs to be included in the evaluation, not every number must be included in the

estimate of the expected value.

When the YjS do not substantially coincide (i.e., when the results from different methods are substantially biased relative to their assigned uncertainties and these biases actually impact

fitness for purpose), there is no universally accepted statistical answer. The approximate

Bayesian method detailed in Levenson et al. provides reasonable values when some independent

knowledge of the method biases is available [80]. However, chemically significant biases among

methods that are putatively equal cast strong doubt on the validity of all results. The most

rigorous procedure is to determine the root cause(s) of the bias and either correct the results or

establish which - if any - of the results are trustworthy.

When resources are insufficient to define fully and account for observed among-method

differences, expert opinion may be used to identify the most reasonable composite value and its

associated 95% confidence interval. While use of subjectively evaluated uncertainty

components is metrologically valid [19], it degrades confidence in the resulting values and thus

in the traceability of the results.

5.2 Measurements of Different Measurands Can Be Summed

When the identity of jV different ICs is known, the identities are all different, and the

amounts of each have been individually assayed, it is simple to:

• transform, if necessary, all measures to consistent units, and

• combine the individual estimates into a single quantitative estimate.

Letting {Y, U^siY)} now represent the estimated values for different components, the

expected value for the combination of all the identified components is just the sum

N

[V]

with an approximate 95% expanded uncertainty of

23
5.3 Measurements of Not Quite the Same or Completely Different Measurands...

Combining collective measurements, such as the sum of wic from all unidentified GC-FID

and LC-ELSD peaks, is less simple. When the different estimates can be assumed to include the

same collections of ICs, they can be averaged as in Section 5.1. When the different collections

can be assumed to be quite different, the estimates should be summed as in Section 5.2. When

the collections can be assumed to include some - but not all - of the same ICs, the combined

estimate should be intermediate between these best- and worst-case possibilities. When the

extent of overlap is difficult to establish, then the worst case should be assumed. If the

difference between the best- and worst-case estimates is significant relative to fitness for

purpose, then the ICs should be further characterized.

Combining amount-of-substance fraction, jcc, and mass fraction, wq, estimates is also not

simple when some of the M\c are unknown. An evaluation using the chemically plausible

extreme values for the M\c (for GC peaks, the lowest and highest M\c compatible with the

observed retention times) may enable transformation to common units. When the assumed

values for any M\c of an unidentified IC significantly affects (relative to fitness for purpose) the

transform values, then the ICs should be further characterized.

5.4 Detection-Limited Quantitative Values

When a critical or suspected IC cannot be confirmed as present using a method having fit- for-purpose sensitivity, an appropriate quantitative statement of the amount of the non-detected

IC, ndIC, in the material is "<LoD", where LoD represents the approximate "limit of detection"

for the IC. The true amount of a particular ndIC in the material, «ndic, is constrained to be

between zero and LoD. The total amount of all ndlCs in the material, Z«ndic> is constrained to be

between zero and the sum of the individual LoDs, 2XoD.

In the purity analysis of elemental RMs, it has been traditional to estimate Z"ndic as equal to

one-half of ZLoD. While this is philosophically incorrect [81], it is perhaps defensibly

conservative for elemental RMs given that there are fewer than 100 elemental ICs. Given the

essentially infinite number of ICs that are plausibly in most organic materials, this estimate for

Syndic will grow without bound as the search for ICs becomes more diligent.

There has been considerable discussion about how best to define LoD [82, 83]. However,

routine LoD values are seldom more than rough estimates of the amount of IC at which "If it were there, I'm pretty sure I would have seen it." Rigorous statistical evaluation of such

"guesstimates" is neither practical nor necessary [83]. If the LoD is defined as the approximate

amount of the IC that would be detectable with «95% confidence, the distribution of confidence

on Hndic can be approximated as an exponential distribution parameterized to have 95% of its area between zero and LoD [81]. Perhaps counter-intuitively, the maximum likelihood estimate

of the location of such an exponential distribution is zero. That is, it is most likely that Z«ndic =

0.

The lower bound to the 95% confidence interval for Z«ndic> £#95(Z"ndic), is zero. The upper

bound, £/Z?95(Z«ndic), can be approximated as the square root of the sum-of-the-squared LoD:

24
[9]

It should be noted that £/Z?95(X«ndic) decreases with the diligence of the search for ICs.

Before the presence of a particular IC is evaluated, the UBgs(lC) (that is, the upper bound to the

range of plausible values) is much larger than LoD. Choosing not to determine whether a given

IC is present at a detectable level does not eliminate the uncertainty in whether it actually is

present. On a less philosophical level, summing in quadrature ensures that £/Z?9s(X«ndic) grows

only slowly with the number of explicitly included ndlCs as long as the LoDs have similar

magnitude.

Under the assumption of exponential distributions for the ndlCs, the expected value for the

sum of the amounts of detected and the non-detected components, Y+ Z^ndic, is just Y, since

Syndic = 0. The uncertainty interval for Y+ Z"ndic is asymmetric. It is defined by the lower and

upper bounds:

LB95 (Y + IWndIC ) = Y - 4u295 {Y) + LB;5 Vnn&c ) =Y-U95 (Y)

UB95 (Y + I«ndIC ) = Y+ ^U295 (Y) + UB295 (lnntSiC )

If both £/2?95(Z«ndic) and Ugs(J) are small relative to fitness for purpose and a symmetric

uncertainty is desired, then the LBgs(Y+ Z«ndic) can be treated as if it were a symmetric

U9s(Y+ Z«ndic)- If UBgsQ^rindic) is less than about 30% of UgsiY), then the £/i?95(X«ndic) will

contribute less than 10% of the combined uncertainty and ignoring the asymmetry will have very

little penalty.

5.5 Dealing with Boundary Violations

The true values for any *c or wc are, by definition, constrained to be no less than 0 and no

greater than 1; i.e., they are constrained to be within the interval [0,1]. However, because of the

simplifying assumption that uncertainties are normally distributed, not infrequently a

{xq, Ugsixc)} or {wc, Ug5(wc)} extends below 0 or greater than 1 (hopefully, not both at the same

time!). Rather than attempting a rigorous evaluation of the true shape of the uncertainty

distribution for all such measurands and combining them in some complicated manner, the

following Bayesian-inspired bias-correction and uncertainty expansion procedure has been

suggested for practical use [83, 84].

• Combine all {xc, Ugsixc)} or {wc, U95(wc)} necessary to get a particular result,

{Y, U95(Y)}, without regard to the [0,1] boundaries.

• If Y-U9s{Y) is less than 0, define an asymmetric interval about Y from LBg5 (Y) = 0 to

UB9s{Y) that will contain the plausible value of Y with about 95% confidence. The

UB9s(Y) is determined by finding the smallest a such that

25
L

N{y> Y>

L

N{y> Y> U95 (Y)

U95 (Y)

J

J

dy

dy

> (1-0.05/ 2) [11]

where N(z,Y,U95 (Y)/2) is the probability density for a normal distribution of mean =

7, standard deviation = Ugs(Y)/2, evaluated over y. (This appears much more

complex than it really is: the renormalization of the standardized normal cumulative

distribution function by its truncated area and subsequent determination of a is easily

accomplished using any table of - or spreadsheet function for - the cumulative

normal.)

• If Y+ U^siY) is greater than 1, define an asymmetric interval about 7 from LB^Y) to

UB<)5(Y) = 1 that will contain the plausible value of Y with about 95% confidence.

The LB9s(Y) is determined by finding the smallest a such that

• If 7 is less than 0, set it to 0; if 7 is greater than 1, set it to 1

• The x95% interval confidence interval about 7 is the asymmetric interval defined by

the lower and upper bounds, LBgs{Y) <Y< UBgsiY). This asymmetric uncertainty

can be reported as r$3£gj [19].

If a direct estimate of Exic or Iwic is available (e.g., by DSC or %GC-FID), then the upper

boundary should be (1 - Lxic) +.t/9s(l - Sxic) or (1 - Ewic) + ^9s(l - Swig) rather than 1.

[12]

26
6. AN APPROACH TO PURITY ASSESSMENT

"... [T]he determination of purity can never be a standardized procedure" [9]. The methods

and rigor of analysis necessary and appropriate for a given neat-material RM depend upon the

physical and chemical properties of the PC and ICs of the material and the purpose(s) for which

the RM will be used.

The following outline presents a general approach to the assessment of purity that should

provide chemically appropriate estimates of the amount of substance for the primary component

and relevant impurity components. In conjunction with the basic requirements discussed in

Section 3, following the spirit of this approach will ensure that the results of a purity assessment

are recognized as sound chemical metrology.

Note that not all of the steps below are necessary or appropriate for all materials. Many of

the measurements required can be accomplished using the same spectral or chromatographic

analyses.

1) Is the material fit for purpose?

a. Examine the material for gross inhomogeneity, discoloration, suspended particulates,

insoluble solids, and other potential problems that can be recognized from visual

inspection and inexpensive assays such as dissolution, filtration, melting- and

boiling-point determinations, refractive index, and mass loss on drying. Is the

material plausibly fit for purpose? If not, can it be made so?

b. Confirm the identity of the PC.

c. If practical, determine 1 - Z*ic by DSC. Is this upper-bound estimate ofxpc

sufficiently large for the material to be fit for purpose?

d. If practical, directly detennine wpc with an instrumentally augmented gravimetric

and/or titrimetric method. If these recognized classical methods are inappropriate,

analyze with qNMR on a high-field instrument or other appropriate direct method.

Is the {wpC, £/95(wpc)} sufficiently large for the material to be fit for purpose?

e. Evaluate any critical ICs using whatever method(s) will ensure that they are not

present at levels that would render the material unfit for purpose. If detected,

evaluate {wic, ^(wic)}; if not detected, estimate UBgs(wic). Are the

{wic, Ugsiwic)} sufficiently small for the material to be fit for purpose?

2) If it is (likely to be) fit for purpose, attempt to identify the ICs.

a. Assay/survey organic impurities (including all organic solvents used in the

preparation or purification of the material).

i. Identify all peaks that appear in

! H- and 13 C-NMR (and19F- and 3I P-NMR if

appropriate) spectra. Based on the identified structures, evaluate

{wic, t/95(wic)} using as many different transitions as practical.

27
ii. Identify all peaks that appear in GC-FID (and other appropriate highsensitivity detection systems such as MS, AED, and EC) chromatograms.

Based on the molecular structures and calculated or measured response

factors, evaluate {w\c, Ugsiwic)} using two or more different optimized but

dissimilar separation systems.

iii. Identify all peaks that appear in LC- UV/Vis absorbance and other

appropriate high sensitivity detection systems such as LC-F1 and LC-electron

capture chromatograms. Based on the molecular structures and calculated or measured response factors, evaluate {wic, Uvsiwic)} using two or more

different optimized but dissimilar separation systems.

b. Assay/survey inorganic impurities.

i. If water is a plausible impurity, determine {wmo, Ugs(wiuo)} with a fit-forpurpose method such as thermogravimetry, Karl Fischer titration, or qNMR.

ii. Determine {wic, ^(wic)} for any suspected inorganic ICs with fit-forpurpose method(s). Ashing has limited sensitivity but may be useful for

estimating a lower bound on total inorganics. High-sensitivity, multielement survey technique(s) such as X-ray fluorescence or ICP-MS should

be used when significant levels of inorganic ICs are anticipated.

c. If not all suspected ICs have been detected, ensure that at least one of the methods

used for general characterization was adequately sensitive for each missing IC. If

necessary, conduct a specific search using adequately sensitive method(s). If

detected, evaluate {wic, Ugs(wic)}; if not detected, estimate UBgsiwic).

3) In light of ICs identified (particularly any contaminant components), evaluate whether the

composition of the material is compatible with the known history and properties of the

material. If it is not compatible, evaluate whether the search for contaminant ICs is

sufficiently complete.

4) Calculate {1 - Zwic, ^95(1 - Zwic)}. Do the confidence intervals overlap with those of the

direct determination of wpc? If they do not overlap and the difference between the direct

and indirect estimates is significant relative to fit-for-purpose total uncertainty, search for

the source(s) of the disagreement and rectify.

5) Once the direct {wPC, ^(wpc)} and indirect {1 - Zwic, £A>5(1 - Zwic)} purity estimates

have been determined to describe the same measurand, combine them as in Section 5.1. The

final value and its 95% confidence interval should be chemically consistent with all

available information.

28
7. CASE STUDIES

The following examples illustrate the general approach to purity assessment advocated in this

document. The CRMs cited here were produced by several different providers and were selected

for discussion partly because their certificates provided sufficient background information and

analytical detail to enable independent evaluation of fitness-for-purpose beyond the scope of

their originally stated need. While all of the CRMs discussed are fit for their original purpose(s),

some have analysis flaws that impact the acceptability of their certification under current

metrological practice. The techniques and extent of provided information may not reflect the

providers' current practice.

7.1 SRM 938 4-Nitrophenol

Purpose: "This [material] is intended primarily for use in calibrating spectrophotometers for

clinical analysis in which 4-nitrophenol is the chromogenic compound that is formed" [85]. The

certified value for this material is a specific absorbance. The material was not intended for use

as an amount-of-substance content CRM.

Component Method Amount ^(Amount) Units

4-nitrophenol Titration with alkali 0.9975 - g/g

2-nitrophenol Paired-ion chromatography O.OOl - g/g

3-nitrophenol Paired-ion chromatography 0.001 - g/g

Water Coulometric Karl Fischer 0.0007 - g/g

Data source: Reference [85]. In addition to these values, information on the general

characterization of 4-nitrophenol from various commercial sources and treatment histories is

given in Ref. 86. The material apparently was also analyzed by DSC and "several LC

separations." However, specific values for the SRM material are not presented. The analyses

were apparently performed between 1977 and 1979.

PC identity confirmation: 'H-,

13 C-, and 15N-NMR.

Heterogeneity: "No significant differences among samples [single samples from 10 different

bottles] were observed with either of these tests [alkali titration and Karl Fischer]." [85] No

sample size is specified.

Material history: Commercially obtained material, purified by twice recrystallizing it from

water, once from dichloromethane, followed by a third recrystallization from water. The

material was dried for several days, then sublimed under specified conditions.

Assessment: While this material was not intended for use as a purity CRM, the procedures

used to prepare and evaluate it are exemplary - had the uncertainties of the various analyses

performed been evaluated. Without these uncertainties, the purity value assigned to this material

is not traceable nor can its fitness for purpose as a purity standard be assessed.

29
7.2 SRM 998 Angiotensin I (Human)

Purpose: "This [material] is certified as a chemical of known purity. It is intended primarily

for use in the calibration and standardization of procedures of the renin assay and as a reference

peptide for amino acid analysis and high-performance liquid chromatography (HPLC)." [87]

Component Method Amount ^(Amount) Units

Angiotensin I Amino acid assay 0.941 0.009 g/g

Acetate 'H-NMR 0.063 0.002 g/g

Peptide impurities LC-UV/Vis2 15,280, 'H-NMR None

Non-peptide impurities LC-UV/Vis2 15,280,

! H-NMR, MS Traces

Data source: Reference [87]. The analyses were performed prior to 1983.

PC identity confirmation: LC-UV/Vis2i5,28o with amino acid assay of all detected peaks.

Heterogeneity: "The calculated coefficient of variation between ampoules was 2.9%." [87]

Material history: Commercially obtained material.

Assessment: The primary method of analysis used for this material was the indirect LCUV/Vis demonstration of a single peptide component with two mobile phases followed by a

standard-additions GC assay of the component amino acids. The amount of the known acetate

counter ion "impurity" was established by ^-NMR. No other non-peptide impurities were

found at any but trace levels using LC, NMR, and MS analyses.

The certificate does not combine the wAmgiotensin 1 value with that obtained from l-wacetate- Using the conventional coverage factor of 2 to expand the stated standard deviations, the

indicated {wamgiotensin, ^(^angiotensin)} is 0939 ± 0- 010-

However, the ability of the LC assay to differentiate peptide impurities closely related to

angiotensin I is discussed in supporting documents but not in the Certificate. A supporting

document suggests that an isomeric form of the material may form during aging at 4 °C; the

certificate does not present evidence for the stability of the material at the recommended storage

temperature of -20 °C. The certificate does not describe the standard used in the standards

addition assay nor the authority for a specific molar absorbance essential to the assay. No direct

assay of total moisture is presented. Lacking complete evaluation of suspected impurities and

without adequate documentation of calibration materials, the purity value assigned to this

material is not traceable.

30
7.3 SRM 910 Sodium Pyruvate

Purpose: "This [material] is certified as a chemical of known purity. It is intended primarily

for use in the calibration and standardization of procedures for pyruvate, lactic dehydrogenase,

and glutamic-pyruvic transaminase determinations in clinical analyses, and for critical evaluation

of the routine working or secondary reference materials used in these procedures." [88]

Component Method Amount u{Amount) Units

Sodium pyruvate LC-UV/Vis254 0.9850 0.0003 g/g

Sodium pyruvate 'H-NMR 0.9883 0.0005 g/g

Certified 0.987 0.002 g/g

Parapyruvate LC-UV/Vis254 0.0102 0.0003 g/g

Parapyruvate 'H-NMR 0.0068 0.0005 g/g

Certified 0.009 0.002 g/g

Methanol ! H-NMR 0.0021 - g/g

Pyruvate oligomers LC-UV/Vis254 <0.005 - g/g

Organic impurities other than

13 C-NMR O.006 - mol/mol

parapyruvate and methanol

Moisture ! H-NMR? 0.0028 - g/g

Water-insoluble matter ? 0.00004 - g/g

Data source: Reference [88]. The analyses were performed prior to 1981.

PC identity confirmation: 'H-NMR, 13 C-NMR, and elemental analysis.

Heterogeneity: "The homogeneity of the SRM, as determined by liquid chromatography and

proton and 13 C NMR, was found to be satisfactory." [88]

Material history: Commercially obtained material.

Assessment: The impurity assessment of this material is exemplary. All ICs detected by LCUV/Vis were characterized by *H- and 13 C-NMR. 13 C-NMR was used to establish an upper

bound on the total amount of uncharacterized organic ICs that is smaller than the uncertainties in

the analysis of the identified components. The stabilities of these impurities and of methanol to

material handling were demonstrated.

The certificate does not combine the various results for the ICs nor compare the Wpymvate

values with an estimate of l-Wparapyruvate-2~>iC. Using the conventional coverage factor of 2 to

expand the stated standard deviations, the indicated {wpyruvate, ^(wpyruvate)} is 0.986 ±0.002.

However, little description is provided on the methods used for moisture, water-insoluble

matter, and elemental composition. More critically, the LC and qNMR methods are incompletely described. While spectroscopic response factors for pyruvate and parapyruvate are presented, there is no description of the materials used in their evaluation. Neither the basic

approach nor sufficient experimental details are specified to establish the fitness-for-purpose of

the qNMR evaluations. Without the requisite auxiliary information, the purity value assigned to

this material is not traceable nor can its fitness for purpose as a purity standard be assessed.

31
7.4 LGC1110 (pp'-Dichlorodiphenyl)dichloroethylene (pp'-DDE)

Purpose: "This material is intended for use in the preparation of solutions for the calibration

of analytical instruments used in pesticide residue and formulation analysis." [89]

Component

1 -Ixic

1 - Iwic

1 - Swic

Water

pp'-DDE

Method

DSC

%area GC-FID

%area LC-UV/Vis248 Karl Fischer

Combined

Amount

0.9974

0.9948

0.9966

0.00010

0.996

^(Amount)

0.0006

0.0004

0.0008

0.00008

0.004

Units

mol/mol

~g/g

?

g/g

g/g

Data source: Reference [90]. Some additional information is supplied in Reference [57]

where this material is used as an example of a neat-substance CRM. The analyses were

performed prior to 1997.

PC identity confirmation: Not stated.

Heterogeneity: "The material was assessed on the basis of six randomly selected 2-mg

portions. The material was judged to be homogenous as the variation between portions was not

significantly different from the analytical variation." [90]

Material history: Commercially obtained material of 99% nominal purity, ground, sieved to

<710 um, and dried under vacuum.

Assessment: With the exception of water, none of the ICs are identified. The low amount of

water and of Ewic observed suggests that the 1 - Ixic DSC results are valid estimates of 1 - ZvvicHowever, the DSC results represent only an upper bound to purity and the LC-UV/Vis results are equivocal without supporting evidence on the nature of the non-water ICs. Thus, the "best"

available evidence is that, with 95% confidence, 0.9948 - 0.0004 < wPP'-dde < 0.9974 + 0.0006

or, equivalently, wPP

'-Dde = 0.9948!JS • since tne DSC *pp'-dde is larger than the %GC-FID

Wpp'-dde, traceability is primarily based upon the validity of the %GC-FID assumptions.

32
7.5 Arsenobetaine Master Material for BCR-626

Purpose: High-purity arsenobetaine was prepared, characterized, and used to prepare the

solution standard BCR-626 [91]. BCR-626 is intended for use as a calibrant in arsenic

speciation studies; the master material used in the preparation of BCR-626 is not available.

Component Method

Organic Iwic

Metal Iwic

Arsenic Iwic

Classical ions ZwiC

'H-NMR, GC-FID, fast atom O.0001

bombardment-MS

ICP-OES, energy dispersive X-ray O.0000

fluorescence

HPLC-ICP-MS <0.0015

Capillary zone electrophoresis with <0.002

UV detection

Amount ^(Amount) Units

g/g

g/g

g/g

•g/g

Total C ICP-OES 0.334 0.003 g/g

Total H ICP-OES 0.0625 0.0007 g/g

Total As ICP-OES 0.418 0.008 g/g

H20 Thermogravimetry 0.023 Not stated g/g

Worst case H2O Expected C - Total C 0.009 Not stated g/g

Arsenobetaine Combined 0.997 0.006 g/g

Data source: Reference [91]. The analyses were performed prior to 1997.

PC identity confirmation: Method of preparation and 'H-NMR.

Heterogeneity: Total As was assayed by ICP-OES in all of the intermediate "mother

solutions" prepared from the master material and in the water-solution CRM units prepared from

the mother solutions. Total analysis repeatability for both materials was 0.6%.

Material history: The material was synthesized and purified according to stated procedures

[91].

Observations: The pilot laboratories made a thorough search for ICs. The material was

recognized as very hygroscopic, but moisture content was not well evaluated in material

protected from ambient humidity. Rather than the authors' focus on just the difference between

the observed and calculated total C, least square minimization of the total C, H, and As results

suggests that {wh2o, ^95(^20)} for the material is {0.007, ±0.017} rather than the stated "worst

case" of 0.009. While better information on water is needed for a believable combination, based

on complete analysis of supplied data the certified value would be better stated as 0.993

Traceability is no stronger than its weakest link; in this case, moisture determination.

33
7.6 BCR 289 2,4'-Dichlorobiphenyl (PCB-8)

Purpose: "... intended mainly for the qualitative and quantitative calibration of analytical

apparatus and methods (determination of retention times, response factors and reference spectra

in chromatographic and spectroscopic analysis) and for the study of biological activity." [92]

Component Method Amount LB95 UB95 Units

Organic Swic 23 determinations by 8 laboratories using 0.0034 0.0025 0.0058 g/g

GC-FID, GC-MS, and LC-UV/Vis.

Tentative identification of ICs consistent

with being other Cb-biphenyl isomers.

Inorganic Iwic Ashing ofHF-digest 0.0003 0.0000 0.0006 g/g

Total Swic Combined 0.0037 0.0025 0.0064 g/g

PCB-8 1- Total Iwic 0.9963 0.9936 0.9975 g/g

Data source: Reference [92]. The analyses were performed prior to 1987.

PC identity confirmation: Method of preparation and IR spectroscopy, melting point, GC

retention time, and X-ray diffraction structure determination.

Heterogeneity: "The between-bottle homogeneity was assessed [using an undefined LC

method] on six vials taken at random . . . [the result] was not significantly different from that of a

replicate analysis of a single solution. No within-bottle study was undertaken because previous

studies have shown that the homogenization process used in the preparative phase was fully

effective." [92]

Material history: The material was synthesized and purified according to published

procedures [92].

Observations: This material was evaluated as part of a multi-laboratory, multi-method

investigation. In aggregate, the large number of differing GC and LC analyses provide

reasonable evidence of a complete analysis. The asymmetric confidence bounds result from the

authors' assumption that the individual organic Swic were log-normally distributed. The results are adequately traceable.

34
7.7 NMIJ CRM 401 1-a o-Xylene

Purpose: ".

. .for use in calibration of analytical instruments and other applications." [93]

C!omnoTiPTit V'V/lllUUllvllL 1V1C 111UU /vmouni C95^/vmouni) units

1 Vv„, /\uiauaiic Laiorinieiry U.UUUUz mol/mol

«-nonane GC-FID/MS 0.0000482 0.0000024 g/g

/^-xylene GC-FID/MS 0.0000394 0.0000038 g/g

m-xylene GC-FID/MS 0 000220 0 000020 cr/cr &&

Isopropylbenzene GC-FID/MS 0.000134 0.000006 g/g

Water Coulometric Karl Fischer 0.000015 0.000004 g/g

Inorganics ICP-MS Not stated Not stated g/g

Consecutive 1 - Xxic Combined 0.999543 0.000022 g/g

o-xylene Combined 0.99940 0.00003 g/g

Data source: Reference [93]. The analyses were performed prior to 2002.

PC identity confirmation: Not stated.

Heterogeneity: %Area GC-FID and water assay by Karl-Fischer: "less than uncertainty of

purity determination" [93].

Material history: Commercially obtained special-grade reagent, dehydrated, double-distilled,

and ampouled into dark brown hard glass under argon.

Observations: Only the calorimetric 1 - Xxic is certified, limiting theformal traceability to

the validity of the adiabatic calorimetric method used. However, all of the observed ICs are compatible with material history and the search for contaminants appears adequate. The

assumption that the 1 - Ijcic = 1 - Iwic is not defended but the IC levels are sufficiently small

that it appears valid. While there is no overlap between the direct {0.99933, ±0.00002} and

consecutive {0.99954, ±0.00002} estimates of 1 - Iwic, the calorimetric estimate is somewhat

smaller and therefore quite believable as an estimate of the true amount of all impurities. The

combined estimate of {0.99940, ±0.00003} provided as a reference value thus unnecessarily

combines the direct and consecutive determinations, but would be validly traceable except for

the uncertainty being too small: application of Equation 6 to the two independent determinations

yields a value of {0.9994, ±0.0003 }

35
7.8 Technical Profenofos

Purpose: Technical-grade 0-(4-bromo-2-chlorophenyl) O-ethyl S-propyl phosphorothioate

(profenofos) insecticide was analyzed as part of a project to demonstrate the utility of qNMR

techniques for the direct determination of chemical purity. The material was not intended nor is

it (unfortunately) available for use as an RM [41].

Component Method Amount ^(Amount) Units

Profenofos 'H-NMR 0.9463 0.0031 g/g

Profenofos 31 P-NMR 0.9461 0.0065 g/g

Data source: Reference [41]. The analyses were performed prior to 2002.

PC identity confirmation:

J H- and 31 -P NMR.

Heterogeneity: Within-batch heterogeneity is included in material uncertainty evaluations.

Material history: Commercially obtained material.

Observations: The qNMR analyses of this material are exemplary. All procedures and

standards are identified, all NMR signals are accounted for, and a complete evaluation of sources of uncertainty is provided. NMR-active ICs are identified and shown to be compatible with the

synthetic route. The indicated {wprofenofos, ^(wprofenofos)} vame of 0.946 ±0.007 is fully

traceable to the qNMR standards used. Material stability, evaluation of the NMR-active ICs, and

a survey for NMR-inactive ICs would be the only additional information required to enable

determination of this material's fitness-for-purpose as an RM.

36
7.9 SRM 911b Cholesterol

Purpose: "This [material] is primarily for use in the calibration and standardization of

procedures for the determination of cholesterol in clinical samples and for routine evaluations of

daily working standards used in these procedures." [94]

Component Method Amount ^(Amount) Units

Direct Lxic U.UUUJ7 moi/iTiui

i—j |— 1111! 1 7-Dehydrocholesterol GC-MS 0.0003 g/g

Campesterol GC-MS 0.0005 g/g

Sitosterol GC-MS 0.0005 g/g

Lathosterol GC-MS <0.0002 g/g

Cholestanol GC-MS <0.0002 g/g

Other steroids GC-MS <0.0001 g/g

Consecutive Ewic GC-MS <0.0018 0.0005 g/g

5,7,9(1 l)-trienes UV/Vis <0.0003 g/g

Organic impurities

! H- and 13 C-NMR None >0.001 g/g

Chlorine NAA 0.000006 0.000001 g/g

Bromine NAA 0.000038 0.000007 g/g

Iodine NAA O.0000001 g/g

Volatiles mass loss on drying 0.00020 0.00001 g/g

Insoluble matter filtration 0.00017 g/g

Ash combustion 0.00006 g/g

Cholesterol Combined 0.998 0.001 g/g

Data source: Reference [94] and supporting Reports of Analysis. The analyses were

performed from 1 986 to 1988.

PC identity confirmation: Multiple direct comparisons (including GC-MS, NMR, melting

point, and optical rotation) with SRM 911a; however, there is no explicit statement on record for

the method of identity confirmation used for SRM 91 la or its precursor SRM 911.

Heterogeneity: No significant heterogeneity, as " determined by GC-MS and DSC" [94].

Material history: Commercially obtained material that had been treated with bromine,

dehalogenated with zinc and recrystallized from methanol to remove companion steroids [94].

Observations: Expected ICs, including inorganic and solvents as well as sterols, were

identified and characterized. One observed steroid IC was quantitatively determined by GC-MS

standard addition; all others were estimated from peak heights relative to the characterized IC.

Contaminant ICs were excluded using NMR, UV/Vis, and infrared spectroscopies, thin-layer

chromatography, and GC-MS. Value assignment was by chemical judgment, regarding the DSC

result as an upper-bound and the sum of the expected ICs as a pessimistic lower-bound. Given

the low IC levels, the implicit assumption that Exic « Zwic appears justified. The resulting

certified purity, while stated with less precision and wider uncertainty than a more mathematical

combination could provide, covers the chemically plausible values and is thus fully traceable.

37
8. OPPORTUNITIES

There is little that is unique about the selection, production, analysis, and certification of neat

materials relative to calibration solution and natural-matrix RMs. While no single chemical

measurement technology is by itself fully "primary", a number of methods can be made "primary

enough" when sufficiently validated for the specific determination and augmented by other

measurements and diligent efforts to identify and quantify all impurity components that impact

the material's fitness for purpose. We conclude that currently available analytical methods can

adequately establish the SI traceability of amount of substance measurements of neat materials.

However, several opportunities for improving traceability infrastructure have been identified.

1) Few of the CRMs that are of potential interest as qNMR comparators are suitably certified as molecular entities. For example, NIST SRM 1071b Triphenyl Phosphate (Standard for

Determination of Phosphorus in Petroleum Products) is certified to be {9.48, ±0.08}

phosphorus "weight percent". This total phosphorus value does not provide a traceable link

for use of this material as a source of (C6H5 )3 P04 for a 'H-,

13 C- and 31 P-qNMR standard. It may be possible to recertify some established neat-material CRMs for use with qNMR with

little or no additional chemical characterization. A number of compounds have been

proposed as qNMR comparators that are not currently available as neat-material CRMs.

However, since qNMR compares nuclei and not molecules, it is likely that only a small suite

of neat-material CRMs need to be recertified or developed to meet the traceability needs of

this measurement technology [41].

2) Methods that directly determine the PC mole fraction, such as %qNMR and %GC, can

provide highly repeatable results relatively quickly and inexpensively. However, there is

little information regarding the quantitative evaluation of the uncertainties in such results.

These uncertainty sources include: the uniformity of the response factor relating signal

intensity to mole fraction for all components, the extent to which the total signal represents

the totality of ICs, and the degree of isolation of signal from the PC from that of the ICs.

General procedures for demonstrating the adequacy of the assumptions and evaluating their

uncertainties would enhance the traceability of the results provided by these direct methods.

3) "Measurement-assisted" QSPRs that combine experimental determination of response

factors for a limited set of structurally diverse compounds plus a few compounds similar to

the desired IC could greatly improve the prediction of chromatographic detector response

factors from molecular structure. Such QSPR predictions would improve the quantitative

utility of %GC and %LC methods when exemplar materials for some ICs are too expensive

or unavailable for experimental response factor determination.

4) A very sensitive, universal mass detector for LC would greatly facilitate the development of

%LC methods for purity determination. IRMS and CRIMS, both of which quantitatively

convert molecules to a set of small polyatomic species and enable characterization of the

relative molecular mass of the original compound through the ratios of characteristic stable

isotopes, may meet this need. LC-CRTMS can be used with conventional mass

spectrometers and enables a more complete evaluation of a molecule's elemental

composition. Neither technique has apparently been developed for use in purity

evaluations.

38
9. ACKNOWLEDGMENTS

The authors thank Robert W. Gerlach (Lockheed Martin Information Technology, Las

Vegas, NV USA) and Stephen L.R. Ellison (LGC, Teddington, UK) for arguing over various

issues concerning the combining of not-quite-the-same values. Their insights have been

invaluable, but the current work does not necessarily reflect all of their views.

We thank Harald Jancke (BAM, Berlin, Germany) for his careful critique of the manuscript

and for his guidance on qNMR techniques. We thank Tareq Saed Al Deen (University of New

South Wales, Sydney, Australia) for making available data from his qNMR analyses of technical

Profenofos.

The authors acknowledge the advice and generally gentle correction of their NIST

colleagues: Richard G. Christensen, Pamela M. Chu, Gary W. Kramer, William A. MacCrehan,

Kenneth W. Pratt, Michele M. Schantz, Katherine S. Sharpless, Robert Q. Thompson, John C.

Travis, and Michael J. Welch. Bob Gerlach and Kathy Sharpless have done much to ensure that

what we've written is what we actually meant.

39
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47


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