1、BSI Standards PublicationBS EN 16847:2016Fertilizers Determinationof complexing agents infertilizers Identificationof heptagluconic acid bychromatographyBS EN 16847:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16847:2016. The UK participation in its prep
2、aration 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 provisions of a contract. Users are responsible for
3、its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 89115 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 an
4、d Strategy Committee on 29 February 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 16847:2016EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16847 January 2016 ICS 65.080 English Version Fertilizers - Determination of complexing agents in fertilizers - Iden
5、tification of heptagluconic acid by chromatography Engrais - 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 Eu
6、ropean Standard was approved by CEN on 17 November 2015. CEN 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 reference
7、s concerning such national standards may be obtained on application 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 mem
8、ber into its own language and notified to the CEN-CENELEC Management 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, Fr
9、ance, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, 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
10、KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 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 EBS EN 16847:2016EN 16847:2016 (E) 2 Contents Page European foreword . 3 1 Scope 4
11、2 Normative references 4 3 Terms and definitions . 4 4 Principle . 4 5 Interferences 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 Pr
12、eparation of the HGA-metal complex sample solution . 6 8.2 Preparation of the 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 Reproduci
13、bility 8 11 Test report . 9 Annex A (informative) Chromatograms of the standard 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-laborat
14、ory test . 14 D.1 Inter-laboratory test . 14 D.2 Test Samples 14 D.3 Inter-laboratory test procedure . 14 D.4 Results and statistical interpretation . 14 Annex E (informative) Complete names of chelating agents . 16 Bibliography . 17 BS EN 16847:2016EN 16847:2016 (E) 3 European foreword This documen
15、t (EN 16847:2016) has been prepared by Technical Committee CEN/TC 260 “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 J
16、uly 2016, and conflicting national standards shall be withdrawn at the 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 ri
17、ghts. This document has been prepared under a mandate given to CEN by 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: Aus
18、tria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, 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, Swed
19、en, Switzerland, Turkey and the United Kingdom. BS EN 16847:2016EN 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
20、 EC fertilizers containing complexed micro-nutrients, which are covered 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 t
21、his document and are indispensable for its application. For dated 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
22、 1: General terms EN 12944-2:1999, Fertilizers and liming materials 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
23、definitions given in EN 12944-1:1999 and EN 12944-2:1999 apply. 4 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 chroma
24、tography. The separation is carried out on an NH2phase bonded to 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
25、preventing the identification/determination of HGA. b) High concentrations 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 corr
26、esponding chelating agents do not interfere since they are separated 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 s
27、cheduled. BS EN 16847:2016EN 16847:2016 (E) 5 d) The presence 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 laborato
28、ry equipment, glassware, and the following: 6.1 Magnetic stirrer. 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;
29、 dp = 5 m 1); d) a NH2guard-column (recommended); e) a UV-Vis 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
30、 0,1 mg. 6.5 Membrane filters. Micro membrane filters resistant 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 comm
31、ercially. This information is given for the convenience of users 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. BS EN 16847:2016EN 16847:2016 (E) 6 7.1 Water, conforming to EN
32、ISO 3696, degassed by boiling before use. 7.2 Sample preparation 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
33、 freshly prepared daily, because of the formation of the 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. Aft
34、er dissolution, dilute to the mark with water and homogenize. 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
35、water and homogenize. Before use, filter the solution through 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 decla
36、red metal content of the product. NOTE Sample preparation 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 port
37、ion mg 10 to 15 300 5 to 10 500 5 1 000 Add 50 ml of sample 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 phos
38、phate to form. BS EN 16847:2016EN 16847:2016 (E) 7 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 sol
39、ution stand overnight. Table 3 Composition of the 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 ha
40、s a molecular mass of 226 g/mol. 8.3 Chromatographic 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 chrom
41、atographic system (6.2). The major peak obtained corresponds 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 a
42、cid. Adjust the attenuation on the integrator, in 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 solution
43、s versus the HGA (acid) concentration (mg/l) in the 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
44、 sample solution corresponding to the complexing agent 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.
45、 The retention times of both isomers differ in less 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 p
46、eak. This effect can be observed in e.g. the copper complex or in mixtures of complexes. BS EN 16847:2016EN 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
47、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 for analysis in milligrams. NOTE In commercial products, the molar ratio HGA: Metal (HGA measured accordi
48、ng this method and the amount of complexed metals determined according to EN 15962, see 2) is typically above 0,5 (see Annex C for further explanation) 2). 10 Precision 10.1 Inter-laboratory test An inter-laboratory test has been carried out in 2014 with nine participating laboratories and three dif
49、ferent samples (one Zn-HGA liquid fertilizer, one Fe-HGA solid fertilizer and one micronutrient mix solid fertilizer). The statistical results are summarized in Annex D. Repeatability and reproducibility were calculated according to ISO 5725-2, see 5. The values derived from this inter-laboratory test might not be applicable to concentration ranges and matrices other than those given in Annex D. Moreover, slight modifications (mainly waiting time in the preparation of the calibration solution) were introduced in the method here pres
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