1、BSI Standards PublicationBS ISO 18643:2016Fertilizers and soil conditioners Determination of biuretcontent of urea-basedfertilizers HPLC methodBS ISO 18643:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 18643:2016. The UK participation in its preparation
2、was entrusted to TechnicalCommittee 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 provisions of a contract. Users are responsible for its corre
3、ct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 82552 1 ICS 65.080 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strateg
4、y Committee on 31 March 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 18643:2016 ISO 2016Fertilizers and soil conditioners Determination of biuret content of urea-based fertilizers HPLC methodEngrais et amendements Dtermination de la teneur en biuret des engra
5、is base dure Mthode HPLCINTERNATIONAL STANDARDISO18643First edition2016-04-01Reference numberISO 18643:2016(E)BS ISO 18643:2016ISO 18643:2016(E)ii ISO 2016 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of th
6、is 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 ISOs member body in t
7、he 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 18643:2016ISO 18643:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Principle 14 Reagents 15 A
8、pparatus . 16 Procedure. 26.1 Preparation of test sample 26.2 Preparation of test solution . 26.3 Preparation of biuret working standard solutions 26.4 HPLC conditions . 26.5 Preparation of standard curve . 36.6 Determination of the biuret content in the test solution 36.7 Calculation and expression
9、 of the results 36.8 Precision . 36.8.1 Ring test . 36.8.2 Repeatability, r 46.8.3 Reproducibility, R . 47 Test report . 4Annex A (informative) Report of international laboratories ring test 5Annex B (informative) Alternative method for determination of biuret in fertilizers 11Bibilography .12 ISO 2
10、016 All rights reserved iiiContents PageBS ISO 18643:2016ISO 18643:2016(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 normally carried out through ISO tec
11、hnical 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 work. ISO collaborates closel
12、y 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 different approval criteria nee
13、ded 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 document may be the subject of patent
14、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.org/patents).Any trade name used in
15、this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers
16、 to Trade (TBT) see the following URL: Foreword - Supplementary information.The committee responsible for this document is ISO/TC 134, Fertilizers and soil conditioners.iv ISO 2016 All rights reservedBS ISO 18643:2016ISO 18643:2016(E)IntroductionBiuret, also known as 2-imidodicarbonic diamide (NH2CO
17、NHCONH2), is one of several by-products formed when molten urea is heated near or above its melting point (132 C) during the manufacturing of urea.12The exact mechanism of biuret damage to different plants is still under investigation, but the harmful effects of high concentrations have been well do
18、cumented, and many regulations/standards concerning the maximum allowed concentrations and/or the analytical methods have been published around the world.1345678Nowadays, there are at least three kinds of analytical methods available for the determination of biuret in fertilizers, including traditio
19、nal spectrophotometric methods,57the atomic absorption spectrophotometric method,8and HPLC methods.251011Recently, HPLC methods have shown superiority over other types of methods, owing to their ability to quantitatively determine biuret content by completely separating biuret from numerous urea-con
20、densates. ISO/TC 134 is well aware of great efforts made by analysts/scientists around the world on separately seeking a uniform, quick and accurate method for the determination of biuret in fertilizers and attempted to unify the HPLC method to the greatest extent herein, based on the preliminary re
21、search by the China, US, and European experts.251011 ISO 2016 All rights reserved vBS ISO 18643:2016BS ISO 18643:2016Fertilizers and soil conditioners Determination of biuret content of urea-based fertilizers HPLC method1 ScopeThis International Standard specifies the test procedure for determinatio
22、n of the biuret content in liquid and solid urea-based fertilizers based on the HPLC method.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies.
23、 For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 3696, Water for analytical laboratory use Specification and test methods3 PrincipleThe biuret content in the fertilizer is extracted by aqueous acetonitrile mobile phase and separated from o
24、ther contents by reversed liquid chromatography on an amino/aminopropyl column. The peak is detected by UV detector attached to the HPLC.4 ReagentsWARNING Acetonitrile is flammable and toxic. Refer to applicable Safety Data Sheet (SDS). The related operations shall be performed in the fume hood. Thi
25、s International Standard does not point out all possible safety problems and the user shall bear the responsibility to take proper safety and health measures and ensure the operations are compliant with the conditions stipulated by the related laws and regulations of the state.Use only water conform
26、ing to grade 3 of ISO 3696.4.1 Acetonitrile, HPLC grade.4.2 Mobile phase, 150 ml water + 850 ml acetonitrile, filtered by 0,22 m membrane as pre-treatment, and ultrasonic degas for 10 min before use.NOTE An alternative method using acetonitrile-free mobile phase is listed in Annex B, with some limit
27、ations on the scope of application.4.3 Biuret stock solutions (0,5 mg/ml = 500 ppm), weigh 0,500 0 g high purity Biuret, dissolved by mobile phase (4.2), and transferred into a 1 l volumetric flask, dilute to volume with mobile phase (4.2) and mix.The biuret of at least 97 % purity and the biuret pu
28、rity claimed on the label shall be based on the biuret content, not on the N-content. There are methods available for the purification procedure of biuret such as those described in AOAC 960.04A(c) or ISO 17322.5 Apparatus5.1 Ordinary laboratory apparatus.INTERNATIONAL STANDARD ISO 18643:2016(E) ISO
29、 2016 All rights reserved 1BS ISO 18643:2016ISO 18643:2016(E)5.2 Ultrasonic bath.5.3 High-performance liquid chromatography instrument, with UV detector.5.4 Microsyringe, 5 l 50 l.5.5 Syringe-driven filter, with organic filter membrane of 0,22 m pores.5.6 Injection loop, volume of 10 l.5.7 Sieve, wi
30、th the aperture size of 0,50 mm.6 Procedure6.1 Preparation of test sampleFor urea fertilizers, simply take 500 g of divided sample as the test portion; for compound fertilizers, take a reduced laboratory sample of 100 g, and grind until it passes through a sieve of aperture size 0,5mm. Mix thoroughl
31、y to homogenize the sample. Place in a clean dry bottle with lid.6.2 Preparation of test solutionDuplicate replicate experiments shall be done for the final determination of result.Weigh 0,1 g 0,5 g test sample (accurately to 0,000 2 g, with biuret content of 1 mg 2 mg ca.) into a 25 ml beaker. Add
32、10 ml mobile phase (4.2) and dissolve using an ultrasonic bath for 10 min. Transfer to a 25 ml volumetric flask and dilute to volume with mobile phase (4.2). Mix thoroughly and leave standing. Filter with a syringe filer to obtain the test solution.6.3 Preparation of biuret working standard solution
33、sAccording to Table 1, pipette 0,00 ml, 0,50 ml, 1,00 ml, 3,00 ml, 5,00 ml and 10,00 ml biuret stock solution (4.3) into six separate 25 ml volumetric flasks. Dilute with respective volumes of mobile phase (4.2) and make up to the mark and mix thoroughly. Filter with 0,22 m organic filter membrane.T
34、able 1 Biuret working standard solutionsVolume of biuret stock solution mlMass of biuret mg0,00a0,000,50 0,251,00 0,503,00 1,505,00 2,5010,00 5,00aBlank solution.6.4 HPLC conditionsRecommended operating conditions of HPLC are listed in Table 2. Other HPLC conditions that can achieve the same separat
35、ion effects may be used.2 ISO 2016 All rights reservedBS ISO 18643:2016ISO 18643:2016(E)Table 2 Recommended operating conditions of HLPCChromatographic columnAmino column or Aminopropyl column,a4,6 mm 250 mm, with 5 m packingFlow rate 1,0 ml/min 1,3 ml/minInject volume 10 lColumn temperature 35 CDet
36、ector wavelength 195 nmaIf the column is new or has not been in service for more than a week, condition the column for 4 h at room temperature with LC-grade isopropanol at a flow rate that will maintain at least 200 bars column back pressure. This is typically 1 ml/min. Wash the column again for 4 h
37、 with 100 % LC-grade acetonitrile at flow rate of 1 ml/min followed by washing with the mobile phase at flow rate of 1 ml/min until a stable base line is achieved.NOTE 1 The best separation condition can be determined according to different equipment and situations.NOTE 2 Depending on the type of ur
38、ea-based fertilizers, other conditions may apply.NOTE 3 Alternative method using acetonitrile-free mobile phase is listed in Annex B.6.5 Preparation of standard curveEnsure that the HPLC apparatus operating conditions are optimized. Successively inject 10 l working standard solution (6.3) and determ
39、ine the series of biuret working standard solution. Each working standard solution shall be determined two times. Draw the standard curve or get the linear regression equation by calculating the average peak areas of the biuret and the corresponding mass.6.6 Determination of the biuret content in th
40、e test solutionDetermine the test solution (6.2) with the same method of the working standard solution, measure the peak area, and calculate the biuret mass in each test solution according to the standard curve or linear regression equation. After completing the determination, first wash the system
41、with mobile phase (4.2) for 30 min, then wash the system with absolute acetonitrile (4.1) for 30 min. Finally, turn off the apparatus according to the operating procedures.6.7 Calculation and expression of the resultsThe mass fraction of biuret (%), w, is calculated as given in Formula (1):wmm=1-310
42、100 (1)wherem1is the mass of biuret, in mg, of the test solution, calculated according to the standard curve or linear regression equation corresponding to the peak areas;m is the mass, in g, of the test portion.The determination result is the arithmetic average of the parallel determination results
43、.6.8 Precision6.8.1 Ring testDetails of ring test on the precision of the method are summarized in Annex A. ISO 2016 All rights reserved 3BS ISO 18643:2016ISO 18643:2016(E)6.8.2 Repeatability, r For all levels, the repeatability limit r is 0,022, with the unit of mass fraction (%).6.8.3 Reproducibil
44、ity, R For all levels, the reproducibility limit R is 0,102, with the unit of mass fraction (%).7 Test reportThe test report shall contain at least the following information:a) all information necessary for the complete identification of the sample;b) test method used with reference to this Internat
45、ional Standard, i.e. ISO 18643:2016;c) test results obtained;d) date of sampling and sampling procedure (if known);e) date when the analysis was finished;f) whether the requirement of the repeatability limit has been fulfilled;g) all operating details not specified in this International Standard, or
46、 regarded as optional, together with details of any incidents occurred when performing the method, which might have influenced the test results.4 ISO 2016 All rights reservedBS ISO 18643:2016ISO 18643:2016(E)Annex A (informative) Report of international laboratories ring testA.1 Generala) Internatio
47、nal laboratories ring tests based on ISO/CD 18643 were conducted between January 2014 and March 2014. Thirteen laboratories participated in the two parallel tests on seven test samples.b) The test method described in ISO/CD 18643 was adopted here for the determination of biuret contents in the ferti
48、lizer samples.c) Seven different types of fertilizer samples were used during the ring test, each with several mean levels. The test samples were sample A-NPK compound fertilizer, sample B-urea formaldehyde complex fertilizer, sample C-urea, sample D-NPK complex fertilizer, sample E-urea ammonium ni
49、trate (UAN) solution, sample F-polymer sulfur coated urea (PSCU), and sample G-urea formaldehyde slow release liquid fertilizer (Trisert1). The biuret contents in all of the seven fertilizer samples lie in the range of 0,09 % 1,01 % (mass fraction).d) The precision of the test results was evaluated based on ISO 5725-2.A.2 Statistical analysis of the test results of biuret contentsA.2.1 Original test resultsThirteen laboratories participated in the determination o
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