1、Designation: D 3598 89 (Reapproved 2003)Standard Test Method forCitrate in Synthetic Detergents1This standard is issued under the fixed designation D 3598; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 appli
3、cable to most detergents containing citrate at aminimum concentration of approximately 5 % (1-8).21.2 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health
4、 practices and determine the applica-bility of regulatory limitations prior to use. Material SafetyData Sheets are available for reagents and materials. Reviewthem for hazards prior to usage.2. Referenced Documents2.1 ASTM Standards:D 501 Test Methods of Sampling and Chemical Analysis ofAlkaline Det
5、ergents3D 1193 Specification for Reagent Water43. Summary of Test Method3.1 This test method employs an enzyme system that isbased upon the selective cleavage of citrate by citrate lyase(citrate oxaloacetate-lyase; EC 4.1.3.6) (1). One of the prod-ucts, oxaloacetate, is reduced to malate by malic de
6、hydroge-nase (L-malate: NAD oxidoreductase; EC 1.1.1.37) with thesimultaneous oxidation of reduced b-nicotinamide adeninedinucleotide to b-nicotinamide adenine dinucleotide, oxidizedform. The course of the reaction is measured spectrophoto-metrically. The decrease in absorbance at 340 nm caused by t
7、heformation of b-nicotinamide adenine dinucleotide, oxidizedform, is directly proportional to the concentration of citrate.4. Interferences4.1 The test method is highly specific for citrate. Otherorganic acids, for example, cisand trans-aconitic, d,l-isocitric,a-ketoglutaric, oxalic, succinic, or ta
8、rtaric 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 metallic ions including zinc andmagnesium will cause inactivation of the enzyme and poten-tially interfere with the te
9、st 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 340 nmunder test conditions.5. Apparatus5.1 Interval Timer.5.2 Micropipet, suitable Eppendorf pipets for dispensin
10、g 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-fluorocarbon stoppers and a minimumvolume of 4 mL.6. Reagents6.1 Purity of ReagentsReagent grade chemicals shall be
11、used 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,where such specifications are available.5Other grades may beused, provided it is first ascertained that the rea
12、gent 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 Detergents, and is the direct responsibility of Subcommittee D12.12 onAnalysis of Soaps and Synthetic Detergents
13、.Current edition approved May 26, 1989. Published July 1989. Originallypublished as D 3598 77 T. Last previous edition D 3598 80 (1988).2The boldface numbers in parentheses refer to the references at the end of thistest method.3Annual Book of ASTM Standards, Vol 15.04.4Annual Book of ASTM Standards,
14、 Vol 11.01.5Reagent 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 LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States
15、 Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to me
16、an Type II reagent waterconforming to Specification D 1193.6.3 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 40 units/mL; for example, 2.0 mL of water is added toa
17、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 icebath for the duration of the analyses and can be used for
18、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 citrate lyase powder should be stored as specified by thesup
19、plier.6.4 Disodium b-Nicotinamide Adenine Dinucleotide, Re-duced Form Solution (0.0032 M)Dissolve 10 mg of diso-diumb -nicotinamide adenine dinucleotide, reduced form,(b-NADH) in 4.0 mL of water. The b-NADH should beapproximately 98 % pure and essentially free of the alphaisomer. The b-NADH solution
20、 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 mL of HCl (spgr 1.19) to 700 mL of water and with
21、 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 example, 1.5mL of water is added to a vial contai
22、ning 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 b-NADH to l-malateand b-NAD per minute at pH 7.5 at 25C. The MDH solutionshould be maintained in an ice bath for the durati
23、on 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 be stored as specified by the supplier.6.8 Tr
24、iethanolamine 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 citrate dihy-drate, accurately weighed, in water and dilu
25、te 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 V10(1)where:CI,II= concentration of trisodi
26、um 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 water.7. Sampling7.1 Collect the sample in acc
27、ordance with Test MethodsD 501.8. 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 water. This is thesample test solution.8.2 Du
28、ring 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 waterblank, standard Solutions I or II, or a samp
29、le test solution.8.4 Pipet 2.0 mL of the triethanolamine buffer solution, 100L of the b-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. Stopper the celland mix by inverting severa
30、l 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 inverting several times. Again do not sh
31、ake 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,II5DASTD2DABC(2)where:DASTD=(A1A2) = decrease in absorbanc
32、e due to thetrisodium citrate dihydrate contentof the standard solution,DAB=(A1A2) = decrease in absorbance for the wa-ter blank, andC = concentration of trisodium citratedihydrate in the standard solution,g/mL.9.2 Calculate the trisodium citrate dihydrate content of thesample as follows:D 3598 89 (
33、2003)2Trisodium citrate dihydrate, % 5DASAMPLE2DABW 3 FAV G3 1.5(3)where:DASAMPLE=(A1A2) = decrease in absorbance due tothe trisodium citrate dihydratecontent of the sample,DAB=(A1A2) = decrease in absorbance for thewater blank,W = sample weight, g, andFAV G=F11 FII /2 = average of the factors calcu
34、-lated for each of the standardsolutions.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 citrate; enzyme cleavage; synth
35、etic detergentsREFERENCES(1) Taraborelli, J. A., and Upton, R. P.,“ Enzymatic Determination ofCitrates in Detergents,” Journal of the American Oil Chemists Society,JAOCA, Vol 52, p. 248.(2) Berka, A., and Hilgard, S., “Bestimmung organischer Stoffe durchOxidation mit Permanganat. IV. die Oxidation v
36、on 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 Chemical Communica-tions, CCCA, Vol 29, 1964, p. 1361
37、.(4) Hartford, C. G., “Rapid Spectrophotometric Method for the Determi-nation of Itaconic, Citric, Aconitic, and Fumaric Acids,” Analyt-icalChemistry, ANCHA, Vol 34, 1962, p. 426.(5) Pucker, G. W., Sherman, C. C., and Vickery, H. B., “A Method toDetermine Small Amounts of Citric Acid in Biological M
38、aterial,”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 Citrate with CitrateLyase,” A
39、nalytical 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 connection with any item mentione
40、din 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 responsible technical committee and
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42、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 Barr Harbor Drive, PO Box C
43、700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).D 3598 89 (2003)3
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