1、ICS 13.060.50Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung Gemeinsamerfassbare Stoffgruppen (Gruppe F) Teil 22: Bestimmung von Glyphosat und Aminomethyl-phosphonsure (AMPA) in Wasser durch Hochleistungs-Flssigkeitschromatographie (HPLC),Nachsulenderivatisierung und Fluore
2、szenzdetektion (F 22)In keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker.ForewordThis standard has been jointly prepared by the Normenausschuss Wasserwesen (Water Practice Standards
3、Committee) and Study Group Wasserchemie (Water Chemistry) of the Gesellschaft Deutscher Chemiker(German Chemists Society) (cf. Explanatory notes).Expert assistance and specialized laboratories will be required to perform the analyses specified in thisstandard. Existing safety instructions shall be o
4、bserved.Depending on the objective of the analysis, a check shall be made on a case-by-case basis as to whetherand to what extent additional boundary conditions will have to be specified.IntroductionGlyphosate is a non-selective leaf herbicide that is generally used in the form of its isopropylammon
5、ium ortrimethylsulfonium salt (glyphosate-trimesium) in various crop protection agents and that can be employed,sometimes along with other active herbicidal ingredients, for weed control in plant production and to removevegetation on road surfaces. Its main metabolite is aminomethylphosphonic acid,
6、which may also be formedby the degradation of other phosphonic acids.CAUTION. Users of this standard should be familiar with standard laboratory practice. It is not claimedthat all the safety problems associated with the use of the standard have been dealt with exhaustivelyhere. It is therefore the
7、responsibility of the user to take appropriate safety precautions and to ensurethat these comply with national regulations.Ref. No. DIN 38407-22 : 2001-10English price group 10 Sales No. 011012.02DEUTSCHE NORM October 200138407-22 No part of this translation may be reproduced without the prior permi
8、ssion 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).Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.German sta
9、ndard methods for the examination of water, waste water and sludgeSubstance group analysis (group F)Part 22: Determination of glyphosate and aminomethylphosphonic acid (AMPA)in water by high performance liquid chromatography (HPLC), postcolumnderivatization and fluorescence detection (F 22)Continued
10、 on pages 2 to 17.Page 2DIN 38407-22 : 2001-101 ScopeThis standard specifies a method of determining glyphosate and its main metabolite aminomethylphosphonicacid (AMPA) (cf. table 1) in drinking water, ground water and surface water in concentrations by mass greaterthan 0,05 g/l. It does not detect
11、these substances when adsorbed on suspended matter.Table 1: Physical and chemical parameters for glyphosate and AMPA 1, 2Trivial name Glyphosate AMPAChemical name N-phosphonomethylglycine Aminomethylphosphonic acidMolar mass 169,08 g/mol 111,04 g/molMelting point 200 C (decomposes) 285 C (decomposes
12、)Solubility in water at 25 C 1,2 g/100 ml 5,8 g/100 ml2 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. These norma-tive references are cited at the appropriate places in the text, and the titles of the publications are listed below.
13、For dated references, subsequent amendments to or revisions of any of these publications apply to thisstandard only when incorporated in it by amendment or revision. For undated references, the latest edition ofthe publication referred to applies.DIN 12242-1 Interchangeable conical ground joints on
14、laboratory glassware Dimensions and toler-ancesDIN 12680-2 Graduated cylinders with ring marks for laboratory useDIN 38402-12 German standard methods for the examination of water, waste water and sludge Gen-eral information (group A) Sampling from stagnant waters (A 12)DIN 38402-13 German standard m
15、ethods for the examination of water, waste water and sludge Gen-eral information (group A) Sampling from aquifers (A 13)DIN 38402-14 German standard methods for the examination of water, waste water and sludge Gen-eral information (group A) Sampling of untreated water and drinking water (A 14)DIN 38
16、402-15 German standard methods for the examination of water, waste water and sludge Gen-eral information (group A) Sampling from flowing waters (A 15)DIN 38402-51 German standard methods for the examination of water, waste water and sludge Gen-eral information (group A) Calibration of analytical met
17、hods, evaluation of analyticalresults and linear calibration functions used to determine the performance characteristicsof analytical methods (A 51)DIN EN ISO 1042 Laboratory glassware One-mark volumetric flasks (ISO 1042 : 1998)DIN EN ISO 4796-2 Laboratory glassware Bottles Part 2: Conical neck bot
18、tles (ISO 4796-2 : 2000)DIN ISO 1773 Laboratory glassware Narrow-necked boiling flasks (ISO 1773 : 1997)3 PrincipleThe water sample is acidified and passed through a cation exchanger to obtain an eluate with higher concen-trations of glyphosate and AMPA. After acidification with hydrochloric acid, t
19、he eluate is purified by filtering itthrough an anion exchanger. Alternatively, if the sensitivity of the fluorescence detector is high enough, theacidified sample is filtered through an adsorbent. In both cases, the filtrate is evaporated to dryness and theresidue is taken up in a buffer solution f
20、or the HPLC analysis 3.Glyphosate and AMPA are separated by isocratic HPLC using a cation exchanger and immediately subjectedto a two-stage postcolumn derivatization in which glyphosate is converted to glycine by oxidation with sodiumhypochlorite in the first step (see equation (1):(1)Glyphosate Gly
21、cinePage 3DIN 38407-22 : 2001-10Secondly, the primary amines glycine and AMPA are derivatized with o-phthaldialdehyde in the presence of 2-mercaptoethanol to produce isoindole derivatives that are detected by fluorescence (see equation (2) 3, 4:Glycine4 Interference4.1 Sampling and sample preparatio
22、nTo avoid interference, collect samples as described in clause 8, taking into account the specifications ofDIN 38402-12 to DIN 38402-15. To prevent blockage of the packing in the columns or cartridges due tosuspended matter, filter the sample before preparation through a glass fibre filter.The prese
23、nce of oxidizing agents in the sample, particularly free chlorine, results in the degradation ofglyphosate and AMPA, which takes place in a few minutes at a concentration of, for example, 0,3 mg/l freechlorine. Chlorinated samples shall therefore be stabilized with sodium thiosulfate.4.2 HPLC analys
24、is4.2.1 Overlapping peaksCompounds that exhibit or suppress fluorescence and have chromatographic properties similar to those of thesubstances to be determined will interfere with the analysis and, depending on their quantity, may affect theaccuracy of the result. Peaks broader than those of the ref
25、erence substances and asymmetrical ones are anindication of overlap. The signal cannot be evaluated in such cases.Normally, fluorescent impurities that may result in poorer integration of the glyphosate peak are eluted at thebeginning of the analysis. Peak integration shall therefore be checked for
26、all the samples, and corrected ifnecessary.4.2.2 Unstable retention timesIf the HPLC pump is working satisfactorily and there are no leaks, continuously increasing retention times inthe chromatographic separation with cation exchangers indicate contamination of the stationary phase withiron(III) ion
27、s that may originate from samples or from the HPLC system. It can be eliminated by rinsing theprecolumn and separating column with about 10 ml of phosphoric acid buffer (as in subclause 7.25).4.2.3 Peak areas not reproducibleDecreasing glyphosate peak areas accompanied by increasing AMPA peak areas
28、may indicate that the concen-tration of the oxidizing agent is too low. In such cases, prepare a new oxidizing solution using fresh sodiumhypochlorite. If even this does not restore the peak areas, there may be other causes, for example a differencebetween the actual reactor temperature and that set
29、, quench effects due to system contamination or a changein the sensitivity of the fluorescence detector.4.2.4 Baseline drift and high background fluorescenceIf the purity of the eluent and reagent solutions is satisfactory, but high background fluorescence or a markedbaseline drift is encountered, t
30、his may indicate that the continuous-flow cell of the fluorescence detector iscontaminated. In such cases, first rinse the cell with isopropanol (e.g. using a syringe), and if backgroundfluorescence is still high, clean the cell with 10 ml each of nitric acid, water and isopropanol.4.2.5 Baseline fl
31、uctuationsPeriodic baseline fluctuations are an indication that high residual pump pulsation is interfering with peakdetection and integration, particularly if the measurement sensitivity of the fluorescence detector is high. Theinterference, which may be due to the HPLC pump or the reagent pumps, c
32、an generally be eliminated byinserting additional pulsation dampeners.5 DesignationDesignation of the method of determining glyphosate and aminomethylphosphonic acid in water by high-performance liquid chromatography, postcolumn derivatization and fluorescence detection (F 22):Method DIN 38407 F 22P
33、age 4DIN 38407-22 : 2001-106 Apparatus6.1 GeneralThe glassware used shall be free of substances that may interfere with the determination of glyphosate andAMPA. They can generally be removed by treating with water containing a surfactant and then rinsing withacetone.The following equipment shall be
34、used.6.2 Narrow-mouth, flat-bottom sampling flask, of nominal capacity 1000 ml, preferably made of brownglass, having an NS 29/32 glass stopper (as in DIN 12242-1) (e.g. ISO 4796-2 1000 NJ laboratory flask).6.3 Borosilicate glass fibre filter, with a fibre diameter of 0,75 m to 1,5 m and free of org
35、anic binders.6.4 pH meter, with electrode, to cover the whole pH range.6.5 Magnetic stirrer, with a PTFE*)-coated magnetic follower having a length of about 40 mm.6.6 Wide-mouth flask, made of brown glass, for storing exchanger material, of nominal capacity 1000 ml,having an NS 60/46 glass stopper (
36、e.g. ISO 4796-2 1000 WJ laboratory flask).6.7 Glass chromatography column, for enriching the sample and purifying the extract, having an internaldiameter of 12 mm and a length of 1000 mm, equipped with a no. 2 porosity glass frit and a PTFE outletstopcock.6.8 Glass wool or glass frits, for covering
37、the column packing.6.9 Graduated cylinders, of nominal capacities 10 ml, 100 ml and 250 ml (e.g. DIN 12680-2 MH 10 gradu-ated cylinders).NOTE: Glass or PTFE flask-mounted dispensers may be used instead of graduated cylinders.6.10 Dropping funnel with pressure equalization, of nominal capacity 250 ml
38、, having an NS 14/23 stopper(as in DIN 12242-1) and PTFE outlet stopcock.6.11 Glass vessel, for collecting eluates, of nominal capacity 50 ml (e.g. ISO 1773 conical flask).6.12 Disposable polypropene or glass syringe, of nominal capacity 20 ml, with Luer cone.6.13 Polypropene or glass cartridge, wit
39、h Luer-lock connection, filled with an adsorbent (as in subclause 7.9).6.14 Glass vessel, for evaporating extracts (e.g. pear-shaped flask), of nominal capacity 25 ml or 100 ml,with an NS 14/23 glass stopper (as in DIN 12242-1).6.15 Equipment for evaporating extracts (e.g. rotary evaporator with vac
40、uum stabilizer and temperature-controlled water bath).6.16 Glass vacuum filtration equipment (e.g. a suction flask), of nominal capacity 1000 ml, and filter funnel,of nominal capacity 250 ml, with PTFE-coated perforated sieve for membrane filter, having a diameter of 47 mm.6.17 Membrane filter (e.g.
41、 of cellulose acetate), having a diameter of 47 mm, for filtering buffer solutions.6.18 Microfilter (e.g. nylon membrane filter), having a pore size of 0,45 m, for clarifying the extracts forHPLC analysis.6.19 Glass sample bottles, of capacity about 2 ml, having an inert closure (e.g. a PTFE-coated
42、septum), forstoring the extracts.6.20 Volumetric flasks, of nominal capacities 10 ml, 100 ml and 1000 ml (e.g. ISO 1042 A 10 C volumetricflasks).6.21 Microlitre syringes, of nominal capacities 50 l, 100 l, 250 l, 500 l and 1000 l.NOTE: Precision piston-operated pipettes with glass or polypropene tip
43、s are also suitable for preparing thereference solutions (as in subclause 7.29).6.22 High-performance liquid chromatograph, designed to suit the intended working range and equippedwith*) PTFE = Polytetrafluoroethylene.Page 5DIN 38407-22 : 2001-10a) a low-pulsation analytical pump, with eluent divert
44、er valve or suitable for gradient elution;b) a manual or automatic sample injection system, for sample volumes of up to 100 l;c) a degassing device (e.g. a helium or vacuum degasser), for degassing eluents;d) column oven or thermostat;e) fluorescence detector, preferably with monochromator on the ex
45、citation or emission side (filtration unitsare also suitable; cf. subclause 9.4.3);f) data evaluation system.Since the standard phases used for the chromatographic separation of glyphosate and AMPA are particularlysensitive to iron(III) ions, all the materials with which the eluent comes into contac
46、t prior to chromatographyshould preferably be free of iron. This applies especially to eluent filters, which must be made of titanium, glassor suitable plastics (e.g. polyether ether ketone (PEEK).6.23 Separating column, having an internal diameter of up to 4,6 mm and a length of up to 250 mm, pref-
47、erably containing a strongly acidic cation exchanger resin based on polystyrene/divinylbenzene, in the potas-sium form, with a precolumn of the same material. The material shall be capable of separating glyphosate andAMPA completely (see figure 1 and table 2).NOTE 1: Strongly basic anion exchangers
48、are also suitable for separating glyphosate and AMPA.NOTE 2: Special HPLC columns for separating glyphosate and AMPA are commercially available.See table 2 for operating conditions.Figure 1: Chromatographic separation of glyphosate and AMPAGlyphosateGlyphosateFluorescence intensityFluorescence inten
49、sityPage 6DIN 38407-22 : 2001-10Table 2: Operating conditions for isocratic separation and postcolumn derivatization (examples)Chromatogram AInjected solution 100 l of 1 pg/l standard solution in potassium dihydrogenphosphate,having a pH value of 2,05.Column Marked AColumn temperature 55 CEluent Potassium dihydrogenphosphate/phosphoric acid buffer solution,having a pH value of 2,05.Flow rate 0,4 ml/minPressure 50 barReagent pump no. 1 Oxidizing solution composed of 950 ml of ready-to-use buffer sol
