ISO_NWIPs-for-Ballot

ANSI ACCREDITED US TAG   ISO TC 34 

OPEN BALLOTS IN ISO TC 34  ISO NWIP DOCUMENTS 

THIS BALLOT CLOSES ON OCTOBER 1, 2019

Form 4: New Work Item Proposal

Circulation date: 2019-07-09 Closing date for voting: 2019-10-01 Proposer

Reference number: ISO/NP 24583 (to be given by Central Secretariat)

ISO/TC 34 N 2078

(e.g. ISO member body or A liaison organization)

JISC

Secretariat AFNOR/ABNT

A proposal for a new work item within the scope of an existing committee shall be submitted to the secretariat of that committee with a copy to the Central Secretariat and, in the case of a subcommittee, a copy to the secretariat of the parent technical committee. Proposals not within the scope of an existing committee shall be submitted to the secretariat of the ISO Technical Management Board. The proposer of a new work item may be a member body of ISO, the secretariat itself, another technical committee or subcommittee, an organization in liaison, the Technical Management Board or one of the advisory groups, or the Secretary-General. The proposal will be circulated to the P-members of the technical committee or subcommittee for voting, and to the O-members for information.

The proposer has considered the guidance given in the Annex C during the preparation of the NWIP. Proposal (to be completed by the proposer)

FORM 4 – New Work Item Proposal Version 02/2018

Title of the proposed deliverable.

Quantitative nuclear magnetic resonance spectroscopy -- Purity determination of organic compounds used for foods and food products -- General requirements French title: English title:

(In the case of an amendment, revision or a new part of an existing document, show the reference number and current title) Scope of the proposed deliverable. This document provides general requirements for the measurement methods with quantitative nuclear magnetic resonance (qNMR), using the nuclear species hydrogen (1H), applied to purity determination of organic compounds used for foods and food products. This document is applicable to determine the purities of food-related substances such as food additives, pesticides, natural toxins, and functional compounds. This document provides general requirements and performance criteria for making the measurement results to ensure to be SI traceable. Purpose and justification of the proposal* Background of the proposal 1) Accurate quantification of food components is important to secure food safety and assurance. Presently, chromatography, such as GC and HPLC is used in majority of legal regulations associated with food. For accurate quantification, it is important to use good standards, such as Certified Reference Material (CRM), which are SI traceable with accurate purity. However, based on using the existing methods such as the mass balance method, the preparation of SI-traceable CRMs has lots of trouble that make us difficult to obtain them. Therefore, we focused on the establishment of qNMR as an accurate and rapid method to produce SI traceable reference materials and standardized it in Japan. The methods that can obtain SI-traceable values are called the primary methods, and include isotopic dilution mass spectrometry, coulometric analysis, gravimetric analysis, titrimetric method, freezing point depression method, etc. At present, our established qNMR has the qualification of the primary ratio method that is one of the primary methods. 2) In 2018, an international summit was held to discuss international standardization of the qNMR (qNMR summit 2018 in Tokyo). The qNMR summit was first held by the United States Pharmacopoeia (USP) in October 2016, followed by German NMI, Federal Institute for Materials Research and Testing (BAM) holding the second summit in March 2017. The qNMR summit 2018 in Tokyo was attended by many experts and practitioners of the qNMR worldwide, and lectures and discussions were held over two days. As a result, a consensus was obtained from all attendees, and it was concluded in Japan that international standardization of the qNMR is inevitable to improve the accuracy of food analysis in future. Through such background, a new proposal was made for TC 34 in Japan. Consider the following: Is there a verified market need for the proposal? What problem does this standard solve? What value will the document bring to end-users? See Annex C of the ISO/IEC Directives part 1 for more information. See the following guidance on justification statements on ISO Connect: https://connect.iso.org/pages/viewpage.action?pageId=27590861

Sustainable Development Goals (SDGs) Goal 3: Good Health and Well-Being for People

FORM 4 – New Work Item Proposal Version 06/2018

Preparatory work

(at a minimum an outline should be included with the proposal)

A draft is attached

An outline is attached

An existing document to serve as initial basis

The proposer or the proposer's organization is prepared to undertake the preparatory work required: Yes No If a draft is attached to this proposal: Please select from one of the following options (note that if no option is selected, the default will be the first option): Draft document will be registered as new project in the committee's work programme (stage 20.00) Draft document can be registered as a Working Draft (WD – stage 20.20) Draft document can be registered as a Committee Draft (CD – stage 30.00) Draft document can be registered as a Draft International Standard (DIS – stage 40.00) If the attached document is copyrighted or includes copyrighted content: The proposer confirms that appropriate permissions have been granted in writing for ISO or IEC to use that copyrighted content. Is this a Management Systems Standard (MSS)?

Yes

No

NOTE: if Yes, the NWIP along with the Justification study (see Annex SL of the Consolidated ISO Supplement) must be sent to the MSS Task Force secretariat (tmb@iso.org) for approval before the NWIP ballot can be launched.

Indication(s) of the preferred type to be produced under the proposal. International Standard

Technical Specification

Publicly Available Specification

Proposed development track

24 months 48 months Note: Good project management is essential to meeting deadlines. A committee may be granted only one extension of up to 9 months for the total project duration (to be approved by the ISO/TMB). 18 months* 36 months

*DIS ballot must be successfully completed within 13 months of the project's registration in order to be elligible for the direct publication process

Draft project plan (as discussed with committee leadership) Proposed date for first meeting: Dates for key milestones: DIS submission Publication 2019-11-28 2021-10-31 2022-10-31 Known patented items (see ISO/IEC Directives, Part 1 for important guidance)

Yes

No

If "Yes", provide full information as annex

FORM 4 – New Work Item Proposal Version 06/2018

Co-ordination of work: To the best of your knowledge, has this or a similar proposal been submitted to another standards development organization?

If “Yes”, please specify which one(s): Yes No

Presently, there are many kinds of ISO standards regarding quantification by using of NMR, but they are no work item and published standard describing requirements regarding the application of qNMR to ensure metrological traceability for the measurement of food related substances. A statement from the proposer as to how the proposed work may relate to or impact on existing work, especially existing ISO and IEC deliverables. The proposer should explain how the work differs from apparently similar work, or explain how duplication and conflict will be minimized. A listing of relevant existing documents at the international, regional and national levels. Related official methods, technical documents, etc. are shown below. 1. General chapter of the United States Pharmacopoeia (USP): Quantitative method is described in the commentary of NMR. 2. European Federation of National Associations of Measurement, Testing and Analytical Laboratories (EUROLAB) issues Guide to NMR Method Development and Validation - Part I: Identification and Quantification. 3. Bureau international des poids et mesures (BIPM) has published ISRD (qNMR Internal Standard Reference Data). 4. Quantitative methods are described in general chapter of NMR in the China Pharmacopoeia. National level documentsJapan Japan’s Standards are listed as follows, these methods are mandatory for standard materials of chemical compounds such as food additives and pharmaceuticals. 1. Japan’s Specifications and Standards for Food Additives (JSFA), under the Food Sanitation Act in Japan. 2. The Japanese Pharmacopoeia (JP) 17th under the Law on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical devices 3. Japanese Industrial Standards (JIS) specifies the standards used for industrial activities in Japan. Please fill out the relevant parts of the table below to identify relevant affected stakeholder categories and how they will each benefit from or be impacted by the proposed deliverable(s).

Benefits/impacts

Examples of organizations / companies to be contacted

Industry and commerce large industry

Confidentially self-made reference material and analysis that is SI- traceable using FT-NMR instrument

- Eurofins - Nestlé Ltd. - PepsiCo, Inc - Unilever N.V.

Industry and commerce SMEs

Confidentially self-made reference material and analysis that is SI- traceable using FT-NMR instrument

-Spectral Service AG -DSM Nutritional Products -Sigma-Aldrich

Government

Supply RM/CRM, especially organic compounds

-JRC -FDA -TFDA -AIST

Consumers

By supplying standard substances, it is possible to obtain foods that safety has been confirmed.

General consumers

FORM 4 – New Work Item Proposal Version 06/2018

Labour

Confidentially self-made reference material and analysis that is SI- traceable using FT-NMR instrument

Users of standard substances such as ISO / 17025 accreditation testing laboratories and quality control field.

Academic and research bodies

Confidentially self-made reference material and analysis that is SI- traceable using FT-NMR instrument

- University of Illinois in Chicago - University of Wuerzburg

Standards application businesses

Accreditation of testing laboratories for standard substance producers

-JAB -A2LA

Non-governmental organizations

Provide and utilize technical information on food safety

International Life Sciences Institute

Other (please specify)

Liaisons: A listing of relevant external international organizations or internal parties (other ISO and/or IEC committees) to be engaged as liaisons in the development of the deliverable(s).

Joint/parallel work: Possible joint/parallel work with:

IEC (please specify committee ID)

CEN (please specify committee ID)

ISO/REMCO, USP

Other (please specify)

A listing of relevant countries which are not already P-members of the committee. USA

Note: The committee secretary shall distribute this NWIP to the countries listed above to see if they wish to participate in this work Proposed Project Leader (name and e-mail address) Dr Naoki SUGIMOTO nsugimot@nihs.go.jp / isoqnmr@qnmr-jp.org Name of the Proposer (include contact information) Hideo Sato (Food and Agricultural Materials Inspection Center (FAMIC) iso_famic@famic.go.jp This proposal will be developed by: An existing Working Group: A new Working Group: (title: ISO/TC 34/WG 24 (see doc N2079)) (Note: establishment of a new WG must be approved by committee resolution)

The TC/SC directly To be determined:

FORM 4 – New Work Item Proposal Version 06/2018

Supplementary information relating to the proposal This proposal relates to a new ISO document This proposal relates to the adoption as an active project of an item currently registered as a Preliminary Work Item This proposal relates to the re-establishment of a cancelled project as an active project Other:

Maintenance agencies and registration authorities This proposal requires the service of a maintenance agency. If yes, please identify the potential candidate:

This proposal requires the service of a registration authority. If yes, please identify the potential candidate:

NOTE: Selection and appointment of the MA or RA is subject to the procedure outlined in the ISO/IEC Directives, Annex G and Annex H, and the RA policy in the ISO Supplement, Annex SN. Annex(es) are included with this proposal (give details) see draft proposal Additional information/question(s)

FORM 4 – New Work Item Proposal Version 06/2018

別添3 For ISO TC 34 Secretariat: JISC

Quantitative nuclear magnetic resonance spectroscopy ― Purity determination of organic compounds used for foods and food products ― General requirements WD/CD/DIS/FDIS stage Warning for WDs and CDs This document is not an ISO International Standard. It is distributed for review and comment. It is subject to change without notice and may not be referred to as an International Standard. Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to provide supporting documentation.

ISO/NP

© ISO 2017 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISO's member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyright@iso.org Web www.iso.org Published in Switzerland.

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Contents

page

Foreword .......................................................................................................................................................................... 4 Introduction..................................................................................................................................................................... 5 1 Scope .................................................................................................................................................................... 6 2 Normative references .................................................................................................................................... 6 3 Terms and definitions.................................................................................................................................... 6 4 Principles ........................................................................................................................................................ 11 4.1 Nuclear magnetic resonance spectroscopy ............................................................................................... 11 4.2 1 H quantitative NMR ( 1 H qNMR).................................................................................................................... 13 5 Apparatus and equipment ........................................................................................................................ 16 5.1 General...................................................................................................................................................................... 16 5.2 Installation conditions and safety ................................................................................................................. 17 5.3 Outline of apparatus configuration............................................................................................................... 19 6 Preparation for 1 H qNMR........................................................................................................................... 20 6.1 General...................................................................................................................................................................... 20 6.2 Samples and reference material preparation with a balance ............................................................ 21 6.3 R eference materials ............................................................................................................................................. 22 6.4 Selection of a deuterated solvent................................................................................................................... 22 6.5 NMR tubes ............................................................................................................................................................... 23 6.6 NMR apparatus optimization .......................................................................................................................... 23 6.7 Weighing of sample and reference materials for qNMR ...................................................................... 23 6.8 Sample solution preparation ........................................................................................................................... 24 6.9 Measurement conditions for 1 H qNMR........................................................................................................ 25 7 Measurement ................................................................................................................................................. 26 7.1 1 H qNMR measurement condition settings ............................................................................................... 26 7.2 Measurement procedure................................................................................................................................... 26 7.3 Saving and processing FID data...................................................................................................................... 26 8 Analysis ............................................................................................................................................................ 27 8.1 General...................................................................................................................................................................... 27 8.2 Calculation............................................................................................................................................................... 27 9 Quality assurance requirements ............................................................................................................ 28 9.1 General...................................................................................................................................................................... 28 9.2 Qualification of the NMR apparatus.............................................................................................................. 28 9.3 System suitability test ........................................................................................................................................ 28 9.4 Ensuring traceability........................................................................................................................................... 29 9.5 Ensuring the reliability of analysis values ................................................................................................. 29 10 Test report ...................................................................................................................................................... 30 Bibliography ................................................................................................................................................................. 31

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Foreword 1 ISO (the International Organization for Standardization) is a worldwide federation of national 2 standards bodies (ISO member bodies). The work of preparing International Standards is normally 3 carried out through ISO technical committees. Each member body interested in a subject for which a 4 technical committee has been established has the right to be represented on that committee. 5 International organizations, governmental and non-governmental, in liaison with ISO, also take part in 6 the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all 7 matters of electrotechnical standardization. 8 The procedures used to develop this document and those intended for its further maintenance are 9 described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the 10 different types of ISO documents should be noted. This document was drafted in accordance with the 11 editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives ). 12 Attention is drawn to the possibility that some of the elements of this document may be the subject of 13 patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of 14 any patent rights identified during the development of the document will be in the Introduction and/or 15 on the ISO list of patent declarations received (see www.iso.org/patents ). 16 Any trade name used in this document is information given for the convenience of users and does not 17 constitute an endorsement. 18 For an explanation on the voluntary nature of standards, the meaning of ISO-specific terms and 19 expressions related to conformity assessment, as well as information about ISO's adherence to the WTO 20 principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary 21 information 22 23 The committee responsible for this document is ISO/TC 34, Food products. 24

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Introduction 25 Quantitative analysis using nuclear magnetic resonance (NMR) spectroscopy has been used since the 26 1960s by researchers in many countries. However, it was not used as a general measuring procedure, 27 since a practical method for such quantitative analysis was not established. 28 Both the NMR equipment and the computers for data processing have dramatically advanced in their 29 performances since around the year 2000, which has made the quantification by NMR possible[see ref. 30 23]. 31 For preparation of this document, the NMR equipment, optimization of a measuring specification, and 32 data analysis and automation have been considered. Research and development have been carried out 33 for reference materials suitable for 1 H nuclei which do not interfere with the quantitative analysis of an 34 analyte, and for a method to obtain an analytical value for organic compounds for which traceability to 35 the International System of Units (SI) has been established. 36 On this basis, the purity and the content of organic compounds can now be measured using a 37 conventional method. This helps obtain rapid results and reliable analytical values for analytes in 38 chemicals, medical supplies, unregulated drugs and foodstuffs [see ref. 2, 3 and 12]. 39

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— Purity

Quantitative nuclear magnetic resonance spectroscopy 40 determination of organic compounds used for foods and food 41 products — General requirements 42 1 Scope 43 This document provides general requirements for the measurement methods with quantitative nuclear 44 magnetic resonance (qNMR), using the nuclear species hydrogen ( 1 H), applied to purity determination 45 of organic compounds used for foods and food products. 46 This document is applicable to determine the purities of food-related substances such as food additives, 47 pesticides, natural toxins, and functional compounds. 48 This document provides general requirements and performance criteria for making the measurement 49 results to ensure to be SI traceable. 50 51 52 2 Normative references 53 The following documents are referred to in the text in such a way that some or all of their content 54 constitutes requirements of this document. For dated references, only the edition cited applies. For 55 undated references, the latest edition of the referenced document (including any amendments) applies. 56 ISO/IEC 17025, General requirements for the competence of testing and calibr tion laboratorie 57 ISO 17034, General requirements for the competence of reference material producers 58 OIML R111-1, 2004, Weights of classes E1, E2, F1, F2, M1, M1–2, M2, M2–3 and M3 ― Part 1: Metrological 59 and technical requirements 60 61 3 Terms and definitions 62 For the purposes of this document, the following terms and definitions apply. 63 ISO and IEC maintain terminological databases for use in standardization at the following addresses: 64 • ISO Online browsing platform: available at http://www.iso.org/ob 65 • IEC Electropedia: available at http://www.electropedia.org/ 66 3.1 67 nuclear magnetic resonance 68 NMR 69 physical phenomenon of absorption of radio frequency radiation and resonance of atomic nuclein a 70 static magnetic field. 71

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Note 1 to entry: The resonance among nuclear spins in a magnetic field is based on the transition between their 72 energy levels, arising from interactions between the spins and the static magnetic field. 73 3.2 74 quantitative NMR 75 qNMR 76 quantitative analysis using NMR spectroscopy 77 3.3 78 1 H quantitative NMR 79 1 H qNMR 80 quantitative NMR spectroscopy using hydrogens ( 1 H) as the observed nucleus 81 3.4 82 Resonance 83 Signal of a certain nucleus in an NMR spectrum 84 3.5 85 Signal 86 resonance peak in an NMR spectrum 87 3.6 88 radio frequency 89 RF 90 electromagnetic radiation with a frequency in the range of 300 Hz to 3 THz 91 Note 1 to entry: The RF used for 1 H NMR is in the range of several tens of to about one thousand MHz 92 93 3.7 94 nuclear spin 95 property describing the magnetic moment of a nucleus 96 Note 1 to entry: It can also be described as the spin magnetic moment of a nucleus. 97 98 3.8 99 static magnetic field 100 magnetic field that does not vary over time 101 3.9 102 external magnetic field 103 magnetic field applied to the NMR apparatus from an outside source 104 3.10 105 gyromagnetic ratio 106 natural constant, unique for each nuclear species, that is the ratio of its magnetic moment to its angular 107 momentum 108 Note 1 to entry: The gyromagnetic ratio reflects the angular frequency of the precession of the atomic nuclei in a 109 magnetic field. 110 3.11 111 relaxation 112 A process of a nucleus returning to a state of thermal equilibrium after it has been excited by radio 113 frequency 114

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115

3.12 relaxation time 116 time constant, required for nucleus to obtain an equilibrium state in a magnetic field and to return to 117 that state after excitation with a radio frequency pulse. 118 3.13 119 chemical shift 120 δ 121 relative difference, in an NMR spectrum, between the resonant frequencies of a nucleus and that of a 122 reference material (conventionally: The 1 H resonance signals of Tetra Methyl Silan areset to zero ppm) 123 Note 1 to entry: Chemical shift is expressed in parts per million (ppm) 124 Note 2 to entry: The chemical shift reference frequency for 1 H NMR is that of TMS, set to δ = 0 ppm. 125 3.14 126 spin-spin coupling 127 J coupling 128 spin coupling 129 effect of covalently-bound nuclear spins on each other, causing splitting of their resonance signals 130 3.15 131 spin-spin coupling constant 132 J coupling constant 133 J value 134 spin coupling constant 135 magnitude of the effect of covalently-bound nuclear spins on each other, expressed in Hz 136 3.16 137 NMR spectrum 138 all NMR resonances acquired within a chemical shift region 139 3.17 140 signal area 141 area under the curve of a resonance 142 3.18 143 pulse flip angle 144 tilt angle of the nuclear magnetization vector, relative to the static magnetic field when applying an RF 145 pulse of specific duration and amplitude in a static magnetic field at thermal equilibrium 146 3.19 147 equilibriummagnetization 148 macroscopic magnetic state of an object when placed in a statice magentic field and left for some time to reach the 149 equilibrium state. 150 151 3.20 152 repetition 153 timeperiod of time from the application of the first pulse of a pulse sequence until the same pulse is 154 applied 155 3.21 156 1 H qNMR spectrum 157 frequency domain NMR spectrum obtained using quantitative NMR conditions with hydrogen ( 1 H) as 158 the observed nucleus 159

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160

3.22 NMR spectrometer 161 spectroscopic apparatus that utilizes the nuclear resonance phenomenon of radio frequency absorption 162 in a magnetic field 163 3.23 164 superconducting magnet 165 electromagnet made from coils of superconducting material, capable of maintaining a permanent flow 166 of electric current and operating at cryogenic temperatures to produce a high magnetic field 167 3.24 168 NMR probe 169 apparatus for transmitting RF pulses to and receiving RF signals from a specimen 170 3.25 171 high-frequency pulse generator 172 apparatus consisting of an oscillator and a pulse generator to produce high-frequency pulses 173 3.26 174 Fourier transform NMR 175 FT-NMR 176 NMR apparatus that uses broadband radio frequency pulses to induce the NMR phenomenon, acquires 177 the radio frequency signal emitted during the relaxation process in the time domain, performs Fourier 178 transformation on the signal to convert it to a frequency-domain spectrum 179 3.27 180 Larmor frequency 181 angular frequency of the Larmor precession, which arises when a nucleus is placed in a magnetic field 182 3.28 183 shim 184 shim coils 185 group of electromagnetic coils used to correct the magnetic field inhomogeneity of a magnet system. 186 187 3.29 188 shimming 189 process of adjusting the current flow in each of the shim coils of an NMR magnet, which are designed to 190 generate a homogeneous static magnetic field in the sample region 191 3.30 192 digital resolution 193 frequency domain digital resolution is defined as spectral width in Hz over number of data points of the 194 real spectrum. 195 196 3.31 197 spectral width 198 width of the spectrum after Fourier transformation, in Hz or ppm 199 3.32 200 decoupling 201 means of eliminating spin coupling 202

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203 204

3.33

free induction decay FID 205 An FID is the time domain signal that results from the application of a radio frequency pulse to a sample 206 in a static magnetic field 207 Note 1 to entry: This non-equilibrium magnetization can be induced by applying an RF pulse of sufficient 208 excitation band width and carrier frequency near the Larmor frequency of the nuclear spins. 209 3.34 210 number of transients 211 number of scans 212 number of times that an FT-NMR experiment is repeatedly co-added to improve the signal-to-noise 213 ratio of the NMR spectrum 214 215 3.35 216 Dummy scan 217 steady state scan 218 transient where radio frequency pulse is performed to establish a steady state but no data is acquired prior to the 219 acquisition of FID signals in FT-NMR 220 221 3.36 222 satellite signal 223 signals arising from fraction of sample containing another NMR active nucleus showing the coupling to 224 this nucleus. 225 226 3.37 227 system suitability test 228 routine testing to confirm the operational status of the analysis system and to verify in advance that the 229 test results of the applicable test method are suitable for the purpose 230 3.38 231 test for required detectability for system suitability test 232 verification that the SNR of the response is sufficiently high at the lowest detection level to ensure 233 reliable integration results. 234 3.39 235 system performance for system suitability test 236 verification that it is possible to specifically analyse the target species to the given specifications 237 3.40 238 system repeatability for system suitability test 239 confirmation of the extent of variation in test results across repeated measurements 240 3.41 241 zero filling 242 insertion of zero values at the end of an FID signal; a means of increasing the digital resolution of FT- 243 NMR spectra 244 3.42 245 baseline correction 246 mathematical procedure used to correct distortion in the baseline of an NMR spectrum 247

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248

3.43 quench 249 phenomenon that occurs when a superconducting magnet cannot maintain the superconducting state 250 and the current flowing in the main coil is converted into heat, causing the liquid helium coolant around 251 the magnet to evaporate rapidly 252 3.44 253 pulse sequence 254 a series of one or more radio frequency pulses applied to the test specimen with defined timing, 255 followed by the acquisition of the FID 256 257 3.45 258 window function 259 mathematical function applied to the FID prior to FT in FT-NMR ; impacts the signal-to-noise 260 ratio, line-width, and other spectral properties 261 3.46 262 reference material for chemical shift 263 reference standard 264 chemical substance or analyte that provides the signal that is used to reference the chemical shift of an 265 NMR spectrum 266 3.47 267 reference material for qNMR 268 calibration standard 269 chemical substance or compound that provides the calibration (quantitative standard) for qNMR 270 analysis 271 3.48 272 deuterated solvent 273 solvent in which some or all of the hydrogen atoms have been replaced with deuterium 274 3.49 275 balance 276 apparatus for weighing a mass 277 Note 1 to entry: Highly accurate digital balances based on the electromagnetic force compensation principle, e.g. 278 an ultramicrobalance, are used to weigh very small quantities of a sample. 279 3.50 280 minimum weight 281 smallest quantity of sample required to weigh in order to achieve a specified relative weighing 282 accuracy 283 [see ref. 6, 9 and 13] 284 4 Principles 285 4.1 Nuclear magnetic resonance spectroscopy 286 Nuclear magnetic resonance (NMR) spectroscopy is an analytical method utilizing the phenomenon that 287 the nuclei in the atoms contained in a sample that is placed in a static magnetic field will resonate at a 288 radio frequency that is unique to each nucleus, and that radio frequency is absorbed when there is a 289 transition from a low-energy nuclear spin state to a high-energy nuclear spin state. The typical target 290 nuclei observed are 1 H, 13 C, 15 N, 19 F, 31 P, 29 Si. 291

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Although all nuclei have a charge, for some nuclear species this charge spins on the nucleus axis. The 292 effect of spinning of the nuclear charge results in the generation of a magnetic moment along the axis. 293 The nuclear moment of this spinning charge, i.e. the nuclear spin ( I ), will have a value of 1/2, 1, 3/2 … 294 n /2 (where n is an integer). A nucleus for which I = 0 means that there is no spin. In quantum mechanics, 295 for a spin I , the number of directions that are possible for the nucleus in a uniform external magnetic 296 field is defined by the expression 2 I + 1. Accordingly, when a nucleus is placed in a magnetic field, the 297 nuclear moment (arising from the spins of the protons and neutrons) is oriented in (2 I + 1) different 298 energy levels. Since I = 1/2 for 1 H and 13 C, there are two energy levels for these nuclei. 299 When a nucleus with gyromagnetic ratio γ is placed in an external magnetic field H 0 , the relationship 300 between the transitions between the energy levels and the applied radio frequency v is given by 301 Formula (1). 302 π2 0 H (1) 303 where 304 is the electromagnetic wave frequency ; 305 is the gyromagnetic ratio; 306 307 H 0 is the external magnetic field. 308 309 When there is resonance (transition between energy states) caused by applying radio waves at 310 frequency , the absorption of radio waves at that frequency (NMR signals) can be observed. Since the 311 absorption coefficient (transition probability) for a nucleus is a constant, and does not depend on the 312 environment, the intensity of the acquired NMR signal is basically proportional to the number of nuclei. 313 The spins that have been shifted to the higher energy level by the transition return to a thermal 314 equilibrium state after a certain period of time. This process is called “relaxation”, and the "relaxation 315 rate" is a measure of the kinetics of the return of magnetisation to its equilibrium value. 316 When a molecule is placed in a magnetic field, the electrons in the molecule shield the nucleus from the 317 external magnetic field. The nuclei within the molecule will be shielded to different extents in each 318 different environment, so the resonant frequency of nuclei will be different in each different 319 environment, which is observed as separate signals. The position of the signal is expressed as the 320 chemical shift, delta ( . 321 Since the resonant frequency changes in proportion to the magnetic field, the chemical shift x is defined 322 by Formula (2), and is a quantity that is independent of the strength of the magnetic field. 323 324 R R R s + v v v x (2) 325 where 326 x is the chemical shift; 327 s is the resonance frequency of the sample nuclei; 328 R is the resonance frequency of the reference nuclei; 329

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R is the chemical shift of the reference nuclei (δ R is zero for TMS). 330 Chemical shifts are normally expressed in parts per million (ppm). The signal position has to be defined 331 with the signal position of the reference compound (reference nucleus). 332 The magnetic field acting on the nuclei in a molecule is not only affected by the surrounding electrons 333 (nuclear shielding), there is also influence from other magnetic nuclei within the molecule (nuclei that 334 have nuclear spin are one such type of magnet themselves). This results in a splitting of the signal due 335 to coupling through the chemical bonds between the nuclear magnets. The total area of the split signals 336 is unchanged, and the method is still quantitative. 337 In first order spectra the spacing between two adjacent components of a split signal is known as the 338 spin-spin coupling constant ( J) . However, in higher order spectra this spacing does not represent the 339 actual J -coupling anymore. Accurate values for J can then be obtained only by a quantum mechanical 340 spectral analysis. J is measured in Hertz (Hz), and is independent of the strength of the external 341 magnetic field. As the number of nuclei that interact with each other increases, the splitting pattern 342 becomes more complex. 343 From the NMR spectrum, the following information can be obtained: chemical shift, spin-spin coupling 344 constant, and signal intensity so on. The information can be used to identify and analyse the structure of 345 a chemical substance, as well as for quantification. 346 Note: The relaxation time cannot necessarily be obtained from every spectrum, so three parameters are typically 347 obtained. The coupling constants are represented in spectra; however, it is sometimes difficult to determine them 348 directly, but peak shape is chemical shift. The intensities of 1 H are also susceptible to relaxation: If a short delay 349 between pulses is used, then 1 H intensities will be affected. The difference is due to 13C's typically longer 350 relaxation times, which requires a much longer delay between pulses for over 99% recovery. 351 One-dimensional 1 H NMR is characterized by the fact that the ratio of the numbers of nuclei in a 352 compound corresponds to the ratio of the signal areas observed on a spectrum when the measurements 353 are made under conditions that ensure the quantitative performance. Furthermore, when one- 354 dimensional 1 H NMR is performed under conditions that ensure quantitative performance, and a 355 reference standard material for qNMR that satisfies the requirements for International System of Units 356 (SI) traceability and has the property that the ratio of the numbers of nuclei in the compound observed 357 on the spectrum corresponds to the signal ratio is used, it is possible to obtain highly-reliable values for 358 the purity and content based on the amount of material (mol). This kind of measurement technique is 359 called 1 H qNMR. 360 4.2 1 H quantitative NMR ( 1 H qNMR) 361 The spectra obtained by proton nuclear magnetic resonance ( 1 H NMR) spectroscopy on a substance 362 dissolved in a solvent have the characteristics described below. These characteristics are utilized to 363 perform analysis of chemical structures. 364 The resonance signals have different chemical shifts that depend on the chemical structure of the 365 analyte. 366 The signals are split by spin-spin interactions arising from the number of 1 H bonded to the adjacent 367 carbon atoms. 368 The signal intensities (areas) are proportional to the number of 1 H resonating at each frequency. 369

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In the 1 H NMR spectra, the proton nuclei ( 1 H) within the same molecule but in different chemical 370 environments are observed as separate signals with different chemical shifts according to the 371 resonance frequencies. The signal intensity S i is given by Formula (3). 372 0 / / i i ) (cos 1 1 sin K 1i r i1 r M e e P VM mN S TT TT (3) 373 where 374 S i is the intensity (area) of a signal; 375 K is a constant; 376 N i is the number of protons; 377 V is the volume of the sample solution; 378 m is the mass of the sample; 379 M is the molar mass of the analyte; 380 P is the purity or content of the analyte; 381 is the excitation pulse angle (flip angle); 382 T 1i is the spin-lattice relaxation time of the nuclei ( 1 H) providing the signal; 383 T r is the repetition time; 384 M 0 is the equilibrium magnetization. 385 The subscript i here indicates each separate signal, and the relaxation times will differ according to the 386 environments of the 1 H nuclei. The signal-to-noise ratio (SNR) is improved by co-adding multiple 387 acquisitions of the spectrum. When this is done if the time between acquisitions uses a T r that is 388 sufficiently longer than the longest relaxation time T 1 in the analyte, the condition of 1 1 1 r / TT e will 389 be satisfied for all signals from the analyte, and the area of a signal will indicate the intensity 390 proportional to the number of resonating nuclei, and Formula (3) can be expressed as Formula (4) 391 below. 392 i k N S (4) 393 where 394 S is the signal area; 395 N i is the number of protons; 396 k is a constant related to the measurement of the sample solution being analysed. 397

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When NMR is used for structural analysis, the detection sensitivity if a priority, so SNR is improved 398 by increasing the number of transients without using an adequately long repetition time. As a result, 399 the ratio of the signal areas to the numbers of 1 H within the molecule is no longer consistently the same. 400 However, under conditions that ensure a quantitative performance, the ratio between the signal areas 401 for resonance signals i and j, showing different chemical shifts within the same molecule, is described 402 by Formula (5). 403 404 where 405 S i is the signal area (intensity) of resonance signal i in the molecule; 406 N i is the number of protons corresponding to resonance signal i in the molecule; 407 S j is the signal area (intensity) of resonance signal j in the molecule; 408 N j is the number of protons corresponding to resonance signal j in the molecule. 409 This quantification relationship can also be applied to the signals derived from two different molecules 410 within the same measurement system. This is because k (Formula 5) applies to all compounds in the 411 sample. 412 In this case, it is assumed that when a sample solution is measured, the excitation pulse angle and the 413 volume of the solution are constant, and do not depend on the target analyte. In such a case, from 414 Formulae (4) and (5), the following Formulae (6), (7) and (8) can be obtained, in which the observed 415 signal area S is proportional to values that depend only on the compound being measured, such as the 416 mass m , molear mass M , and content or purity P of the analyte. 417 j i j i N N S S (5)

S S

k k

N N

c c

418 419

(6) (7) (8)

a

a

a

s

s

s

a S S c N N

c

s

a

s

s

a

© ISO 2017 – All rights reserved 420 421 where 422 P a is the content or purity of the analyte; 423 P s is the purity of the reference material for qNMR; 424 S a is the signal area of the analyte; 425 S s is the signal area of the reference material for qNMR; 426 s P a s s a a s s a S S P a m m M M N N

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k is the constant related to the measurement of the sample solution being analysed; 427 N a is the number of protons in the analyte; 428 N s is the number of protons in the reference material for qNMR; 429 c a is the molar concentration of the analyte; 430 c s is the molar concentration of reference material for qNMR; 431 M a is the molar mass of the analyte; 432 M s is the molar mass of the reference material for qNMR; 433 m a is the mass of the analyte compound; 434 m s is the mass of the reference material for qNMR. 435 Although there are some conditions that must be met, such as no interaction (such as reactions) 436 between the molecules in the solution, and the existence of separated signals with different chemical 437 shifts, Formula (4) indicates that it is possible to measure the content or purity of the analyte compound 438 by performing 1 H qNMR measurements under conditions ensuring quantitative performance if there is 439 a reference material for qNMR with a known purity. In other words, if a material with an accurate purity 440 is provided as a reference, if we have a reference material for qNMR with a known molar mass , it is 441 possible to determine the content or purity of other analyte substances within the same solution 442 measured at the same time. Specifically, if the purity value assigned to the reference material for qNMR 443 has been established in a manner traceable to the SI and the procedure used for the implementation of 444 the qNMR method has been appropriately validated, the method can be used to establish the SI- 445 traceability of the result for the content or purity of the analyte 446 The six key procedures for obtaining precise analysis results with 1 H qNMR are: 447 - Precisely weigh the sample and calibrant materials 448 - Ensure the analyte contained in the sample and calibrant dissolve completely. 449 - Acquire the data under conditions that are suitable for quantification 450 - Select a signal for quantification that does not include impurity signals[see ref. 14] 451 - Process the spectrum properly, phase and baseline correct 452 - Integrate sample and reference peaks correctly 453 NOTE If a signal for quantification is overlapped to impurities, some evaluation methods such as line fitting is 454 effective for eliminating the impurities from signals [see ref. 17]. 455 5 Apparatus and equipment 456 5.1 General 457 The applicable nuclear magnetic resonance apparatus (NMR apparatus) is the type that uses a pulse 458 Fourier transforms (FT) method. 459

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460 461

5.2 Installation conditions and safety

5.2.1 Installation room 5.2.1.1 Size 462 The installation room shall be large enough to provide the space required for the installation and 463 operation of the apparatus, as well as the space needed to manage any stray magnetic field. 464 Furthermore, if it is not possible to reserve a control zone outside the room, the room shall be large 465 enough to completely contain the stray magnetic field of the superconducting magnet within the 466 specified values. 467 NOTE The magnitude of the stray magnetic field differs according to the magnet size and type and distance from 468 the magnetic solenoid/core. Stray field affects all directions and impacts upper and lower rooms as well as 469 adjacent horizontally. 470 5.2.1.2 Ceiling height 471 The room shall have a specified minimum ceiling height in order to perform tasks such as installing the 472 apparatus and refilling the helium tanks. 473 NOTE The required height differs according to the magnet size and type. 474 5.2.1.3 Floor 475 A floor that is not subjected to any significant vibration is preferred. The floor shall also satisfy the 476 specified loading and strength requirements. 477 5.2.2 Air conditioning and ventilation facilities 478 The NMR apparatus should be installed in an air-conditioned room. 479 When the superconducting magnet is installed, several cubic metres of helium and nitrogen gas will be 480 released inside the room. To prevent oxygen deficiency in the room, exhaust fans with suitable 481 ventilation capacity and performance shall be installed. These fans should be selected in accordance 482 with the installation instructions for the NMR apparatus. 483 Furthermore, if the magnetic field is accidentally disrupted or quenched, a large amount of helium gas 484 and nitrogen gas will be released from the superconducting magnet. For this reason, quench ducts to 485 the exhaust ports of the superconducting magnet may need to be installed (See Figure 2). Safety 486 measures should be taken to appropriately handle this release of gas, such as equipping the room with 487 an oxygen concentration meter and adequate ventilation fans. 488 Typical installation conditions for a general 400 MHz NMR apparatus are 489 floor vibration of ≤0,2 cm/s 2 (Gal), 490 floor strength to withstand a load of ≥600 kg,/m 2 491 air temperature range from 17 °C to 27 °C (fluctuation within ±1 °C/h), and 492 relative humidity ≤70 % (see Figure 1). 493 NOTE1 Since the NMR apparatus utilizes a superconducting magnet, warning indications about the strong 494 magnet fields generated should be posted at the entrance to the room and on the superconducting magnet, and 495 unauthorized persons shall not be allowed to enter the room. 496

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