1、Designation: D3598 89 (Reapproved 2016)Standard Test Method forCitrate in Synthetic Detergents1This standard is issued under the fixed designation D3598; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A
2、number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the enzymatic determination ofcitrate in both liquid and solid synthetic detergents. The testmethod is applicab
3、le to most detergents containing citrate at aminimum concentration of approximately 5 % (1-8).21.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concerns, if any,
4、associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Material SafetyData Sheets are available for reagents and materials. Reviewthem for hazards pri
5、or to usage.2. Referenced Documents2.1 ASTM Standards:3D501 Test Methods of Sampling and Chemical Analysis ofAlkaline DetergentsD1193 Specification for Reagent Water3. Summary of Test Method3.1 This test method employs an enzyme system that isbased upon the selective cleavage of citrate by citrate l
6、yase(citrate oxaloacetate-lyase; EC 4.1.3.6) (1). One of theproducts, oxaloacetate, is reduced to malate by malic dehydro-genase (L-malate: NAD oxidoreductase; EC 1.1.1.37) with thesimultaneous oxidation of reduced -nicotinamide adeninedinucleotide to -nicotinamide adenine dinucleotide, oxidizedform
7、 The course of the reaction is measured spectrophoto-metrically. The decrease in absorbance at 340 nm caused by theformation of -nicotinamide adenine dinucleotide, oxidizedform, is directly proportional to the concentration of citrate.4. Interferences4.1 The test method is highly specific for citra
8、te. Otherorganic acids, for example, cisand trans-aconitic, d,l-isocitric,-ketoglutaric, oxalic, succinic, or tartaric acids, do not inter-fere.4.2 Although low levels of zinc or magnesium, or both, arerequired as an activator for the enzyme citrate lyase, exces-sively high levels of divalent metall
9、ic ions including zinc andmagnesium will cause inactivation of the enzyme and poten-tially interfere with the test method (7).4.3 The test method is not applicable to those detergentscontaining components with excessive absorptivity at 340 nmsuch that ultraviolet measurements are inappropriate at 34
10、0 nmunder test conditions.5. Apparatus5.1 Interval Timer.5.2 Micropipet, suitable Eppendorf pipets for dispensing 10and 100-L volumes and with disposable tips.5.3 Spectrophotometer, suitable for measuring ultravioletabsorbance at 340 nm and equipped with 1-cm matched quartzcells with tapered TFE-flu
11、orocarbon stoppers and a minimumvolume of 4 mL.6. Reagents6.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society
12、where such specifications are available.4Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.1This test method is under the jurisdiction of ASTM Committee D12 on Soapsand Other
13、Detergents and is the direct responsibility of Subcommittee D12.12 onAnalysis and Specifications of Soaps, Synthetics, Detergents and their Components.Current edition approved July 1, 2016. Published August 2016. Originallyapproved in 1977 as D3598 77 T. Last previous edition approved in 2009 asD359
14、8 89 (2009). DOI: 10.1520/D3598-89R16.2The boldface numbers in parentheses refer to the references at the end of thistest method.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume informati
15、on, refer to the standards Document Summary page onthe ASTM website.4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemic
16、als, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.2 Purity of WaterUnless otherwise
17、 indicated, referencesto water shall be understood to mean Type II reagent waterconforming to Specification D1193.6.3 Citrate Lyase Solution (40 units/mL)Add sufficientcold water to a vial of citrate lyase containing a premeasuredweight of enzyme protein such that the resulting solution willcontain
18、40 units/mL; for example, 2.0 mL of water is added toa vial containing 5 mg of enzyme protein with an activity of 16units/mg of enzyme protein. One unit of activity will convert1.0 mol of citrate to oxaloacetate per minute at pH 7.6 at25C. The citrate lyase solution should be maintained in an icebat
19、h for the duration of the analyses and can be used for 5 daysif refrigerated. Citrate lyase (EC 4.1.3.6) from Aerobacteraerogenes is commercially available as a lyophilized powdercontaining approximately 24 % citrate lyase, 24 % albumin,48 % saccharose and 4 % magnesium sulfate (MgSO47H2O).The citra
20、te lyase powder should be stored as specified by thesupplier.6.4 Disodium -Nicotinamide Adenine Dinucleotide, Re-duced Form Solution (0.0032 M)Dissolve 10 mg ofdisodium -nicotinamide adenine dinucleotide, reduced form,(-NADH) in 4.0 mL of water. The -NADH should beapproximately 98 % pure and essenti
21、ally free of the alphaisomer. The -NADH solution should be protected from lightand maintained in an ice bath for the duration of the analyses.The solution should be prepared fresh daily.6.5 Hydrochloric Acid (sp gr 1.19)Concentrated hydro-chloric acid (HCl).6.6 Hydrochloric Acid (1 N)Slowly add 85 m
22、L of HCl (spgr 1.19) to 700 mL of water and with mixing dilute to 1 L withwater.6.7 Malic Dehydrogenase Solution (2500 units/mL)Addsufficient cold water to a vial of malic dehydrogenase (MDH)suspension containing a premeasured volume such that theresulting solution will contain 2500 units/mL; for ex
23、ample, 1.5mL of water is added to a vial containing 5 mg of enzymeprotein in 0.5 mL of suspension with an activity of 1000 Munits/mg of enzyme protein. One micromolar unit of activitywill convert 1.0 mol of oxaloacetate and -NADH to l-malateand -NAD per minute at pH 7.5 at 25C. The MDH solutionshoul
24、d be maintained in an ice bath for the duration of theanalyses and can be used for 5 days if refrigerated. MDH (EC1.1.1.37) from Porcine heart is commercially available as asuspension in 2.8 M ammonium sulfate solution, pH 6. TheMDH suspension should contain 0.01 % transaminase activ-ity and should
25、be stored as specified by the supplier.6.8 Triethanolamine Buffer Solution (0.1 M, pH 7.6)Dilute 6.65 mL of triethanolamine in approximately 250 mL ofwater. Adjust to pH 7.6 with 1 N HCl.6.9 Trisodium Citrate Dihydrate Standard Solutions I andIIDissolve approximately 150 mg of trisodium citratedihyd
26、rate, accurately weighed, in water and dilute to 100 mL.Dilute 2.0 and 4.0-mL aliquots of this solution each to 100 mLwith water. These are the standard Solutions I and II, respec-tively. Calculate the actual concentration of trisodium citratedihydrate in each standard solution as follows:CI,II5S 3
27、V10(1)where:CI,II= concentration of trisodium citrate dihydrate in thestandard Solutions I or II, g/mL,S = standard weight of TSC, mg, andV = volume taken for the final dilution, mL.Prepare all solutions fresh daily.6.10 Zinc Chloride Solution (0.003 M)Dissolve 41 mg ofzinc chloride in 100 mL of wat
28、er.7. Sampling7.1 Collect the sample in accordance with Test MethodsD501.8. Procedure8.1 Dissolve an accurately weighed detergent sampleequivalent to approximately 300 mg of trisodium citratedihydrate in distilled water and dilute to 200 mL. Dilute a 3.0mL aliquot of this solution to 100 mL with wat
29、er. This is thesample test solution.8.2 During the following steps, use the appropriate micropi-pet for the 10 and 100-L volumes, replacing the tip after eachaddition. Standard volumetric pipets can be used for the 1.0and 2.0-mL additions.8.3 Into a 1-cm quartz cell, pipet 1.0 mL of either a waterbl
30、ank, standard Solutions I or II, or a sample test solution.8.4 Pipet 2.0 mL of the triethanolamine buffer solution, 100L of the -NADH solution, and 100 L of the zinc chloridesolution into the cell.8.5 Pipet 10 L of the MDH solution below the liquidsurface in the cell and start the interval timer. St
31、opper the celland mix by inverting several times. Do not shake the cell so asto cause foaming.8.6 After 2.0 min, measure the absorbance (A1) at 340 nmversus water.8.7 After an additional 1.0 min, pipet 10 L of the citratelyase solution below the liquid surface in the cell. Stopper thecell and mix by
32、 inverting several times. Again do not shake thecell too vigorously.8.8 After an additional 3.0 min, measure the absorbance(A2) at 340 nm versus water.9. Calculation9.1 Calculate the trisodium citrate dihydrate standard factor(FI,II) for each of the standard solutions as follows:FI,II5ASTD2 ABC(2)wh
33、ere:ASTD=(A1A2) = decrease in absorbance due to the tri-sodium citrate dihydrate content of thestandard solution,AB=(A1A2) = decrease in absorbance for the waterblank, andD3598 89 (2016)2C = concentration of trisodium citrate di-hydrate in the standard solution,g/mL.9.2 Calculate the trisodium citra
34、te dihydrate content of thesample as follows:Trisodium citrate dihydrate, % 5ASAMPLE2 ABW 3 FAVG31.5(3)where:ASAMPLE=(A1A2) = decrease in absorbance due to thetrisodium citrate dihydrate contentof the sample,AB=(A1A2) = decrease in absorbance for the wa-ter blank,W = sample weight, g, andFAVG=F11FII
35、2= average of the factors calculatedfor each of the standard solutions.10. Precision and Bias10.1 Under the most favorable conditions the precision maybe expressed as follows:So5 0.02X (4)where:So= single-operator precision, % w/w, andX = trisodium citrate dihydrate content, % w w.11. Keywords11.1 c
36、itrate; enzyme cleavage; synthetic detergentsREFERENCES(1) Taraborelli, J. A., and Upton, R. P.,“ Enzymatic Determination ofCitrates in Detergents,” Journal of the American Oil ChemistsSociety, JAOCA, Vol 52, p. 248.(2) Berka, A., and Hilgard, S., “Bestimmung organischer Stoffe durchOxidation mit Pe
37、rmanganat. IV. die Oxidation von Milch, Apfel,Zitron und Salicylsaure,” Mickrochim Acta, Vol 174, 1966.(3) Sucha, I., and Volka, K., “Anwendung von Zitratkomplexen mit Ionendes Systems Fe+3/Fe+2bei der Massanalytischen Bestimmung vonZitronensauren.” Collection of Czechoslovak ChemicalCommunications,
38、 CCCA, Vol 29, 1964, p. 1361.(4) Hartford, C. G.,“Rapid Spectrophotometric Method for the Determi-nation of Itaconic, Citric, Aconitic, and Fumaric Acids,” AnalyticalChemistry, ANCHA, Vol 34, 1962, p. 426.(5) Pucker, G. W., Sherman, C. C., and Vickery, H. B., “A Method toDetermine Small Amounts of C
39、itric Acid in Biological Material,”Journal of Biological Chemistry, JBCHA, Vol 113, 1936, p. 235.(6) Dagley, S., in Methods of Enzymatic Analysis, edited by Bergmeyer,H. U., Verlag Chemie Weinheim and Academic Press, New York, NY,1965, p. 313.(7) Moellering, J., and Gruber, W., “Determination of Cit
40、rate with CitrateLyase,” Analytical Biochemistry, ANBCA, Vol 17, 1966, p. 369.(8) Bergmeyer, H. U., Methods of Enzymatic Analysis, Verlag ChemieWeinheim and Academic Press, New York, NY, p. 38, 1965.ASTM International takes no position respecting the validity of any patent rights asserted in connect
41、ion with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsibl
42、e technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful cons
43、ideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 B
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