1、May 2016 English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 65.080!%SM;“2484224www.din.deDIN EN
2、16847Fertilizers Determination of complexing agents in fertilizers Identification of heptagluconic acid by chromatography;English version EN 16847:2016,English translation of DIN EN 16847:2016-05Dngemittel Bestimmung von Komplexbildnern in Dngemitteln Identifikation von Heptaglukonsure mit Chromatog
3、raphie;Englische Fassung EN 16847:2016,Englische bersetzung von DIN EN 16847:2016-05Engrais Dtermination des agents complexants dans les engrais Identification de lacide heptagluconique par chromatographie;Version anglaise EN 16847:2016,Traduction anglaise de DIN EN 16847:2016-05www.beuth.deDTransla
4、tion by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.Document comprises 19 pages 04.16 DIN EN 16847:2016-05 2 A comma is used as the decimal marker. National foreword This document (EN 16847:2016) has been prepared by Technical Committee CEN/TC
5、260 “Fertilizers and liming materials” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-03-02 AA Dngemittel. Th
6、e DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determinatio
7、n of repeatability and reproducibility of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16847 January 2016 ICS 65.080 English Version Fertilizers - Determination of complexing agents in fertilizers - Identification of heptagluconic acid by chromatography Engrais
8、- Dtermination des agents complexants dans les engrais - Identification de lacide heptagluconique par chromatographie Dngemittel - Bestimmung von Komplexbildnern in Dngemitteln - Identifikation von Heptaglukonsure mit Chromatographie This European Standard was approved by CEN on 17 November 2015. CE
9、N members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on app
10、lication to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC M
11、anagement Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, La
12、tvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Ma
13、rnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16847:2016 EEN 16847:2016 (E) 2 Contents Page European foreword . 3 1 Scope 4 2 Normative references 4 3 Terms and definitions . 4 4 Principle . 4 5 Inte
14、rferences 4 6 Apparatus . 5 7 Reagents . 5 7.1 Water, 6 7.2 Sample preparation solvent. . 6 7.3 HGA stock solution, . 6 7.4 Eluent A: ortho-phosphoric acid, . 6 7.5 Eluent B: acetonitrile (HPLC-grade). 6 8 Procedure. 6 8.1 Preparation of the HGA-metal complex sample solution . 6 8.2 Preparation of t
15、he calibration solutions 7 8.3 Chromatographic analysis . 7 9 Calculation of the heptagluconic acid content and expression of the results . 8 10 Precision . 8 10.1 Inter-laboratory test 8 10.2 Repeatability . 8 10.3 Reproducibility 8 11 Test report . 9 Annex A (informative) Chromatograms of the stan
16、dard and a typical sample solution . 10 Annex B (informative) Absorption spectra of the HGA 12 Annex C (informative) Calculation of the molar ratio HGA:Metal . 13 Annex D (informative) Statistical results of the inter-laboratory test . 14 D.1 Inter-laboratory test . 14 D.2 Test Samples 14 D.3 Inter-
17、laboratory test procedure . 14 D.4 Results and statistical interpretation . 14 Annex E (informative) Complete names of chelating agents . 16 Bibliography . 17 DIN EN 16847:2016-05 EN 16847:2016 (E) 3 European foreword This document (EN 16847:2016) has been prepared by Technical Committee CEN/TC 260
18、“Fertilizers and liming materials”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by July 2016, and conflicting national standards shall be withdrawn at the
19、 latest by July 2016. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by
20、the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Est
21、onia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16847:2016-05 E
22、N 16847:2016 (E) 4 1 Scope This European Standard specifies a chromatographic method which allows the identification of heptagluconic acid (HGA) in fertilizers containing heptagluconic acid metal complexes. This method is applicable to EC fertilizers containing complexed micro-nutrients, which are c
23、overed by Regulation (EC) No 2003/2003 1. NOTE For the complete names of the chelating agents mentioned in this document, see Annex E. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated
24、 references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 12944-1:1999, Fertilizers and liming materials and soil improvers Vocabulary Part 1: General terms EN 12944-2:1999, Fertilizers and liming materia
25、ls and soil improvers Vocabulary Part 2: Terms relating to fertilizers EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696) 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 12944-1:1999 and EN 12944-2:1999 apply. 4
26、 Principle The method is based on demetalation with phosphoric acid of the micronutrient HGA complex present in an aqueous solution of the sample. The complexing agent is then identified and determined by high-performance liquid chromatography. The separation is carried out on an NH2phase bonded to
27、silica column and an aqueous solution of phosphoric acid and acetonitrile as eluent. The detection is based on UV photometry at 210 nm. 5 Interferences a) High concentrations of phosphate in the sample solution can create a large peak preventing the identification/determination of HGA. b) High conce
28、ntrations of chloride, sulfate and nitrate do not interfere in the identification/determination of the complexing agent. c) The presence of the chelates of EDDHSA, o,oEDDHA, o,oEDDHMA, EDTA, DTPA, CDTA, HEEDTA, IDHA as well as the corresponding chelating agents do not interfere since they are separa
29、ted from HGA. These substances can be detected in the chromatogram by the appearance of a peak at larger retention times. Therefore, the presence of these kinds of substances shall be taken into account when successive injections are scheduled. DIN EN 16847:2016-05 EN 16847:2016 (E) 5 d) The presenc
30、e of gluconic acid does interfere in the determination of the complexing agent. e) The presence of aspartic acid, humic substances and lignosulfonic acid may interfere with the identification/determination of HGA. 6 Apparatus Usual laboratory equipment, glassware, and the following: 6.1 Magnetic sti
31、rrer. 6.2 Chromatograph, equipped with: a) an isocratic pump delivering the eluent at a flow rate of 1 ml/min; b) an injection valve with a 20 l injection loop or equivalent; c) a NH2column; internal diameter: 4,6 mm; column length: 250 mm; dp = 5 m 1); d) a NH2guard-column (recommended); e) a UV-Vi
32、s detector with a 210 nm-filter; f) an integrator. 6.3 Chromatographic conditions, according to Table 1. Table 1 Chromatographic conditions Flow rate Eluent A (7.4) % Eluent B (7.5) % 1 ml/min 75 25 6.4 Balance, Balance, with an accuracy of 0,1 mg. 6.5 Membrane filters. Micro membrane filters resist
33、ant to aqueous solutions, with porosity of respectively 0,45 m and 0,2 m. 7 Reagents Use only reagents of recognized analytical grade. 1) Phenosphere NH2 80A 5 m 2504,6 mm or equivalent. This is an example of suitable product available commercially. This information is given for the convenience of u
34、sers of this European Standard and does not constitute an endorsement by CEN of this product. Equivalent products can be used if they can be shown to lead to the same results. DIN EN 16847:2016-05 EN 16847:2016 (E) 6 7.1 Water, conforming to EN ISO 3696, degassed by boiling before use. 7.2 Sample pr
35、eparation solvent. Add to 800 ml of water, 2 ml of ortho-phosphoric acid 85 % and 25 ml of methanol in a 1 l volumetric flask. Dilute to the mark with water and homogenize. 7.3 HGA stock solution, c(HGAacid) = 19 893 mg/l. This solution shall be freshly prepared daily, because of the formation of th
36、e corresponding lactone if it let standing for a long period of time. Weigh to an accuracy of 0,1 mg about 2 500 mg of the heptagluconic acid, sodium salt dihydrate (CAS # 10094-62-9), c 99 %, add 50 ml of water in a 100 ml volumetric flask. After dissolution, dilute to the mark with water and homog
37、enize. 7.4 Eluent A: ortho-phosphoric acid, c(H3PO4) = 30 mmol/l and methanol. Add to 800 ml of water, 2 ml of ortho-phosphoric acid 85 % (mass concentration) and 25 ml of methanol (HPLC grade) in a 1 l volumetric flask. Dilute to the mark with water and homogenize. Before use, filter the solution t
38、hrough a 0,45 m membrane filter (6.5). 7.5 Eluent B: acetonitrile (HPLC-grade). 8 Procedure 8.1 Preparation of the HGA-metal complex sample solution The mass of the test portion to be used to prepare the sample solution is dependent on the declared metal content of the product. NOTE Sample preparati
39、on may be conducted according to EN 1482-2, see 6. Weigh into a 150 ml beaker, approximately the amount of sample specified in Table 2, to an accuracy of 0,1 mg: Table 2 Amount of sample Declared metal content % (mass fraction) Mass of test portion mg 10 to 15 300 5 to 10 500 5 1 000 Add 50 ml of sa
40、mple preparation solvent (7.2) and dissolve it with a magnetic stirrer (6.1) during 5 min. Make up to volume in a 100 ml volumetric flask with sample preparation solvent (7.2). Let the solution stand overnight in darkness to allow the metal phosphate to form. DIN EN 16847:2016-05 EN 16847:2016 (E) 7
41、 8.2 Preparation of the calibration solutions Pipette a volume (V ml) (see Table 3) of the HGA stock solution (7.3) in six 100 ml volumetric flasks respectively. Make up to volume with the sample preparation solution (7.2) and homogenize. Let the solution stand overnight. Table 3 Composition of the
42、calibration solutions Solution V ml Concentration of HGA (acid) mg HGA/l 1 1 199 2 2 398 3 6 1 194 4 8 1 591 5 10 1 989 6 16 3 183 NOTE The molecular mass of heptagluconic acid, sodium salt dihydrate corresponds to 284 g/mol, whereas the acid form has a molecular mass of 226 g/mol. 8.3 Chromatograph
43、ic analysis Immediately before injection, all calibration and sample solutions shall be filtered through a 0,2 m membrane filter (6.5). After stabilization of the chromatographic conditions (6.3), inject the calibration solutions (8.2) into the chromatographic system (6.2). The major peak obtained c
44、orresponds to heptagluconic acid. NOTE 1 Since the calibration solutions are not freshly prepared (see 8.2), two defined peaks may appear in the chromatograms, one tentatively assigned to the lactone and the other corresponding to the heptagluconic acid. Adjust the attenuation on the integrator, in
45、order to obtain a suitable range for the HGA peak from the standard solution. A typical chromatogram is given in Figure A.1. Measure the retention time. Draw the calibration curve with the value of the chromatographic peak of the calibration solutions versus the HGA (acid) concentration (mg/l) in th
46、e standards. Inject the sample solution (8.1). Identify the complexing agent by the retention time of the obtained peaks, and if diode array detector is used, confirm it with its UV-visible spectrum (see Annex B). Measure the area of the peak for the sample solution corresponding to the complexing a
47、gent and determine the concentration in (mg/l) using the calibration graph. See Annex A for integration considerations. NOTE 2 Heptagluconic acid can co-exist in two different isomers: alpha and beta. Both isomers can be found in commercial products. The retention times of both isomers differ in les
48、s than 0,3 min and they can be distinguished by two separated peaks depending on the type of column used. NOTE 3 The first part of some chromatograms could present a set of peaks that can disturb dramatically the measurement of the value of the HGA peak. This effect can be observed in e.g. the coppe
49、r complex or in mixtures of complexes. DIN EN 16847:2016-05 EN 16847:2016 (E) 8 9 Calculation of the heptagluconic acid content and expression of the results Calculate the heptagluconic acid (Mw = 226 g/mol) content, wHGA, in the fertilizer, expressed as mass fraction in percent, according to Formula (1). 10HGA=Mw(1) where is the concentration of HGA in milligrams per litre determined with the calibration graph; M is the mass of the sample taken
