1、BS ISO 19746:2017Determination of urea contentin urea-based fertilizers byhigh performance liquidchromatography (HPLC)BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS ISO 19746:2017 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of
2、ISO 19746:2017.The UK participation in its preparation was entrusted to Technical Committee CII/37, Fertilisers and related chemicals.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provi
3、sions of a contract. Users are responsible for its correct application. The British Standards Institution 2017.Published by BSI Standards Limited 2017ISBN 978 0 580 87120 7 ICS 65.080Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published
4、under the authority of the Standards Policy and Strategy Committee on 31 March 2017.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 19746:2017 ISO 2017Determination of urea content in urea-based fertilizers by high performance liquid chromatography (HPLC)Dtermination
5、 de la teneur en ure dans les engrais base dure par chromatographie liquide haute performance (CLHP)INTERNATIONAL STANDARDISO19746First edition2017-02Reference numberISO 19746:2017(E)BS ISO 19746:2017ISO 19746:2017(E)ii ISO 2017 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2017, Published in
6、SwitzerlandAll 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 b
7、e requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 19746:2017ISO 19746:2017(E)Foreword ivIntr
8、oduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principles . 15 Reagents 16 Apparatus . 27 Procedures 27.1 Preparation of test sample 27.2 Preparation of the test solution 27.3 Preparation of urea working standard solution . 37.4 HPLC conditions . 37.5 Preparation of standa
9、rd curve . 37.6 Determination of the urea content in the test solution 47.7 Calculation and expression of results 47.8 Precision . 47.8.1 Repeatability, r . 47.8.2 Reproducibility, R . 47.9 Test report . 4Annex A (informative) Examples of the retention times of triuret, biuret, urea and some other u
10、reaforms 5Bibliography 8 ISO 2017 All rights reserved iiiContents PageBS ISO 19746:2017ISO 19746:2017(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normal
11、ly carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the
12、 work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the d
13、ifferent approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).Attention is drawn to the possibility that some of the elements of this docum
14、ent may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www .iso .o
15、rg/ patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as informati
16、on about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www .iso .org/ iso/ foreword .html.This document was prepared by Technical Committee ISO/TC 134, Fertilizers and soil conditioners.iv ISO 2017 All rights reservedB
17、S ISO 19746:2017ISO 19746:2017(E)IntroductionUrea is the most widely used source of nitrogen fertilizers worldwide and is used in a variety of forms such as pure urea, in combination with other nutrients, in complex fertilizers, and as reacted or modified ureas2.Due to the rapid hydrolysis of urea i
18、n the environment, especially when applied at the soil surface3, efforts have been made to modify urea to slow down this loss process. Slow release nitrogen fertilizers such as methylene urea compounds, controlled release N fertilizers such as sulfur-coated urea (SCU) or polymer-coated urea (PCU), a
19、nd stabilized N fertilizers containing additives (urease inhibitors and nitrification inhibitors) are examples of products aimed at containing the rapid hydrolyses of urea in the soil.Accurate determination of urea in urea-based fertilizers is desirable for regulatory and product quality purposes. T
20、his is especially true for those fertilizers in which the urea content is physically or chemically modified. Most of these modified fertilizers contain some amounts of free and unreacted urea which is readily available N and therefore could not be accounted as part of the slow or controlled release
21、N component5. ISO 2017 All rights reserved vBS ISO 19746:2017BS ISO 19746:2017Determination of urea content in urea-based fertilizers by high performance liquid chromatography (HPLC)1 ScopeThis document specifies the test procedure for determining the urea content in urea-based fertilizers, includin
22、g urea, urea aldehydes methylene urea fertilizers, isobutylene diurea (IBDU), crotonylidene diurea (CDU), urea triazone fertilizers, urea ammonium nitrate (UAN), sulfur- and polymer-coated urea (SCU and PCU), as well as compound fertilizers containing urea. The method is based on High Performance Li
23、quid Chromatography (HPLC).The proposed method is an extension of the AOAC Official Method 2003.14 which was collaboratively studied for the “Determination of Urea in Water-Soluble Urea-Formaldehyde Fertilizer Products and in Aqueous Urea Solutions” in 2003. The method was published in the Journal o
24、f AOAC in 20044 and was granted the First Action in 2003 and the Final Action in 2008.This method also applies to the determination of biuret content in urea containing fertilizer with the results published in the J. AOAC in 20145. This method was adopted by the International Organization for Standa
25、rdization (ISO) as a Committee Draft (ISO/CD 18643) in 2014, and after review and the Ring Test Analyses6.NOTE This HPLC method can also be utilized to analyse Crotonylidene diurea (CDU) and Isobutylidene diurea (IBDU) contents within those above-mentioned fertilizers, in addition to EN 157051.2 Nor
26、mative referencesThe following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any a
27、mendments) applies.ISO 3696:1995, Water for analytical laboratory use Specification and test methods3 Terms and definitionsNo terms and definitions are listed in this document.ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: avail
28、able at h t t p :/ www .electropedia .org/ ISO Online browsing platform: available at h t t p :/ www .iso .org/ obp4 PrinciplesThe urea content in urea-based fertilizer is extracted by aqueous acetonitrile mobile phase and separated from other contents by High Performance liquid chromatography (HPLC
29、) on an aminopropyl column. The urea peak is detected by a UV detector attached to the HPLC.5 ReagentsWARNING Acetonitrile is flammable and toxic. The related operations shall be performed in a laboratory fume hood. This document does not point out all possible safety problems, and the user shall be
30、ar the responsibility to take proper safety and health measures, and ensure the INTERNATIONAL STANDARD ISO 19746:2017(E) ISO 2017 All rights reserved 1BS ISO 19746:2017ISO 19746:2017(E)operations are compliant with the conditions stipulated by the related laws and regulations of the state.Use only w
31、ater conforming to grade 3 of ISO 3696:1995.5.1 Acetonitrile, LC grade.5.2 Mobile phase, 150 ml water + 850 ml acetonitrile. Filter the mobile phase solution using a 0,22 micron membrane and degas with nitrogen gas for 10 min as a pre-treatment before use.5.3 Urea standard solutions, (0,5 mg/ml = 50
32、0 ppm). Weigh 0,5 g of high purity urea, dissolve in mobile phase (5.2) and transfer into a 1 l volumetric flask. Dilute the solution to volume with mobile phase (5.2) and mix thoroughly.6 ApparatusOrdinary laboratory apparatus and the following equipment/instruments:6.1 Ultrasonic bath.6.2 High per
33、formance liquid chromatograph, with UV detector.6.3 Microsyringe, 5 l50 l.6.4 Syringe-driven Filter, with organic filter membrane of 0,22 m pores.6.5 Injection loop, volume of 10 l.6.6 Sieve, with the aperture size of 0,50 mm.6.7 2,00 micron filter paper.7 Procedures7.1 Preparation of test sample7.1
34、.1 For dry urea fertilizers, simply take 500 g of a divided sample as the test sample. For compound fertilizers, take a reduced lab sample of 100 g, grinding until it passes through a sieve of aperture size 0,5 mm, and mix thoroughly for reasons of homogeneity. Place the sample in a clean and dry bo
35、ttle with lid. 7.1.2 For liquid urea fertilizers, take 100 ml of a homogeneous solution. Filter any particulate through 2,0 micron filter paper.For all of these samples, use ultrasonic mixing for 10 min to assure complete dissolution. Filter to remove any undissolved portion through a 2,0 micron fil
36、ter paper.7.2 Preparation of the test solutionA minimum of two replicate experiments shall be done for the analysis of the urea content in fertilizer samples.2 ISO 2017 All rights reservedBS ISO 19746:2017ISO 19746:2017(E)Weigh 0,1 g0,5 g of the dry or the liquid test sample (accurately to 0,000 2 g
37、, with urea content of 1 mg2 mg ca.) into a 25 ml beaker, add 10 ml of the mobile phase (5.2) and dissolve using an ultrasonic bath for 10 min. Transfer the sample to a 25 ml volumetric flask, dilute to volume with the mobile phase (5.2), mix thoroughly and leave standing. Filter with a syringe filt
38、er to obtain the test solution.7.3 Preparation of urea working standard solutionAccording to Table 1, pipette 0,00 ml, 0,50 ml, 1,00 ml, 3,00 ml, 5,00 ml and 10,00 ml of the urea standard solution (5.3) into six separate 25 ml volumetric flasks respectively. Dilute with the mobile phase (5.2) and ma
39、ke up to the mark, then mix thoroughly. Finally, filter each solution through 0,22 micron organic filter membranes.Table 1 Preparation of urea working standard solutionVolume of urea standard solution mlMass of urea mg0,00a0,000,50 0,251,00 0,503,00 1,505,00 2,5010,00 5,00aBlank solution.7.4 HPLC co
40、nditionsRecommended operating conditions for the HPLC method are listed in Table 2. Other HPLC conditions that will achieve the same separation efficiency may be used.Table 2 Recommended operating conditions for the HPLC methodChromatographic columnaAminopropyl column RP-8c,dFlow rateb1,0 ml/min 1,0
41、 ml/minInject volume 10 l 20 lColumn temperature 35 C 35 CDetector wavelength 195 nm 200 nmaExamples of Aminopropyl columns are: Phenomenex Spherex-NH2column, 260 mm 4,6 mm, Part No.00G-0017-E0 and ThermoFisher APS-2 HYPERSIL 250 mm 4,6 mm, with 5 um particle size (Part No.30705-254630).bFlow rate i
42、s a convenient parameter to optimize for specific products, i.e. 1,0 ml/min or 1,3 ml/min. See Annex A for retention times utilizing these two different flow rates (1,0 ml vs. 1,3 ml/min for triazone solutions).cFor the complex triazone liquids, the aminopropyl system has to be used to avoid interfe
43、rences.dColumn used was a LiChroSpher RP-8, 250 mm 4 mm, 5 um.The best separate conditions can be determined according to different equipment and climate conditions.7.5 Preparation of standard curveReferring to the apparatus operation condition (Table 2), adjust the HPLC apparatus to the best operat
44、ing conditions. Inject 10 l of the working standard solution (5.3) and analyse to determine the concentration of the urea in the standard. Each working standard solution shall be determined in a ISO 2017 All rights reserved 3BS ISO 19746:2017ISO 19746:2017(E)minimum of two replicates. Draw the stand
45、ard curve or calculate the linear regression equation using the average peak areas of the urea chromatographic signal and their corresponding mass.7.6 Determination of the urea content in the test solutionDetermine the urea content of each test solution (5.3) using the same method used for the deter
46、mination of the concentrations of the working standard solutions, measure the peak area, and calculate the urea mass in each test solution according to the standard curve or the linear regression equation. After completing the determination, first wash the chromatographic system with mobile phase (5
47、.2) for 30 min, followed by absolute acetonitrile (5.1) for 30 min. Finally, turn off the apparatus according to the operating procedures.7.7 Calculation and expression of resultsThe mass fraction of urea (%), w, is calculated as follows:wmm=1310100 (1)wherem1is the mass of urea of the test solution
48、, in mg, calculated according to the standard curve or the linear regression equation corresponding to the peak areas;m is the mass of the test portion, in g.The arithmetic average of the results shall be calculated and reported.7.8 Precision7.8.1 Repeatability, r 7.8.2 Reproducibility, R NOTE The p
49、recision and other statistical data are left blank at this time. These statistical data have been reported for the AOAC collaborative studies for the determination of urea5and for the ISO biuret ring test studies6.7.9 Test reportThe test report shall contain at least the following information:a) all information necessary for the complete identification of the test samples;b) test method used with reference to this document, i.e. ISO 19746;c) test results obtained;d) date of sampling and sampling pr
