ASTM D3598-89(2003)
(Test Method)Standard Test Method for Citrate in Synthetic Detergents
Standard Test Method for Citrate in Synthetic Detergents
ABSTRACT
This test method covers the enzymatic determination of citrate in both liquid and solid synthetic detergents. The test method is applicable to most detergents containing citrate at a minimum concentration of approximately 5 %. The apparatus to be used in the determination of citrate shall includes interval timer, micropipet, and spectrophotometer. An enzyme system that is based upon the selective cleavage of citrate by citrate lyase (citrate oxaloacetate-lyase; EC 4.1.3.6) shall be employed. One of the products, oxaloacetate, shall be reduced to malate by malic dehydrogenase (L-malate: NAD oxidoreductase; EC 1.1.1.37) with the simultaneous oxidation of reduced (beta)-nicotinamide adenine dinucleotide to (beta)-nicotinamide adenine dinucleotide, oxidized form. The course of the reaction shall be measured spectrophotometrically. The decrease in absorbance at 340 nm caused by the formation of (beta)-nicotinamide adenine dinucleotide, oxidized form, is directly proportional to the concentration of citrate.
SCOPE
1.1 This test method covers the enzymatic determination of citrate in both liquid and solid synthetic detergents. The test method is applicable to most detergents containing citrate at a minimum concentration of approximately 5 % (1-8).
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Material Safety Data Sheets are available for reagents and materials. Review them for hazards prior to usage.
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Designation:D3598–89(Reapproved 2003)
Standard Test Method for
Citrate in Synthetic Detergents
This standard is issued under the fixed designation D3598; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 4. Interferences
1.1 This test method covers the enzymatic determination of 4.1 The test method is highly specific for citrate. Other
citrate in both liquid and solid synthetic detergents. The test organic acids, for example, cisand trans-aconitic, d,l-isocitric,
method is applicable to most detergents containing citrate at a a-ketoglutaric, oxalic, succinic, or tartaric acids, do not inter-
minimum concentration of approximately 5 % (1-8). fere.
1.2 This standard does not purport to address all of the 4.2 Although low levels of zinc or magnesium, or both, are
safety concerns, if any, associated with its use. It is the required as an activator for the enzyme citrate lyase, exces-
responsibility of the user of this standard to establish appro- sively high levels of divalent metallic ions including zinc and
priate safety and health practices and determine the applica- magnesium will cause inactivation of the enzyme and poten-
bility of regulatory limitations prior to use. Material Safety tially interfere with the test method (7).
Data Sheets are available for reagents and materials. Review 4.3 The test method is not applicable to those detergents
them for hazards prior to usage. containing components with excessive absorptivity at 340 nm
such that ultraviolet measurements are inappropriate at 340 nm
2. Referenced Documents
under test conditions.
2.1 ASTM Standards:
5. Apparatus
D501 Test Methods of Sampling and Chemical Analysis of
Alkaline Detergents 5.1 Interval Timer.
D1193 Specification for Reagent Water 5.2 Micropipet, suitable Eppendorf pipets for dispensing 10
and 100-µL volumes and with disposable tips.
3. Summary of Test Method
5.3 Spectrophotometer, suitable for measuring ultraviolet
3.1 This test method employs an enzyme system that is absorbance at 340 nm and equipped with 1-cm matched quartz
based upon the selective cleavage of citrate by citrate lyase cells with tapered TFE-fluorocarbon stoppers and a minimum
(citrate oxaloacetate-lyase; EC 4.1.3.6) (1). One of the prod-
volume of 4 mL.
ucts, oxaloacetate, is reduced to malate by malic dehydroge-
6. Reagents
nase (L-malate: NAD oxidoreductase; EC 1.1.1.37) with the
simultaneous oxidation of reduced b-nicotinamide adenine 6.1 Purity of Reagents—Reagent grade chemicals shall be
dinucleotide to b-nicotinamide adenine dinucleotide, oxidized used in all tests. Unless otherwise indicated, it is intended that
form. The course of the reaction is measured spectrophoto- all reagents shall conform to the specifications of the Commit-
metrically.Thedecreaseinabsorbanceat340nmcausedbythe tee onAnalytical Reagents of theAmerican Chemical Society,
formation of b-nicotinamide adenine dinucleotide, oxidized where such specifications are available. Other grades may be
form, is directly proportional to the concentration of citrate. used, provided it is first ascertained that the reagent is of
sufficiently high purity to permit its use without lessening the
accuracy of the determination.
This test method is under the jurisdiction of ASTM Committee D12 on Soaps
and Other Detergents, and is the direct responsibility of Subcommittee D12.12 on
Analysis of Soaps and Synthetic Detergents.
Current edition approved May 26, 1989. Published July 1989. Originally
published as D3598 – 77 T. Last previous edition D3598 – 80 (1988). DOI: Reagent Chemicals, American Chemical Society Specifications, American
10.1520/D3598-89R03. Chemical Society, Washington, DC. For suggestions on the testing of reagents not
The boldface numbers in parentheses refer to the references at the end of this listed by the American Chemical Society, see Analar Standards for Laboratory
test method. Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Annual Book of ASTM Standards, Vol 15.04. and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
Annual Book of ASTM Standards, Vol 11.01. MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D3598–89 (2003)
6.2 PurityofWater—Unless otherwise indicated, references
where:
to water shall be understood to mean Type II reagent water
C = concentration of trisodium citrate dihydrate in the
I,II
conforming to Specification D1193.
standard Solutions I or II,µ g/mL,
S = standard weight of TSC, mg, and
6.3 Citrate Lyase Solution (40 units/mL)—Add sufficient
V = volume taken for the final dilution, mL.
cold water to a vial of citrate lyase containing a premeasured
Prepare all solutions fresh daily.
weight of enzyme protein such that the resulting solution will
contain 40 units/mL; for example, 2.0 mL of water is added to 6.10 Zinc Chloride Solution (0.003 M)—Dissolve 41 mg of
zinc chloride in 100 mL of water.
a vial containing 5 mg of enzyme protein with an activity of 16
units/mg of enzyme protein. One unit of activity will convert
7. Sampling
1.0 µmol of citrate to oxaloacetate per minute at pH 7.6 at
25°C. The citrate lyase solution should be maintained in an ice
7.1 Collect the sample in accordance with Test Methods
bath for the duration of the analyses and can be used for 5 days
D501.
if refrigerated. Citrate lyase (EC 4.1.3.6) from Aerobacter
aerogenes is commercially available as a lyophilized powder
8. Procedure
containing approximately 24 % citrate lyase, 24 % albumin,
8.1 Dissolve an accurately weighed detergent sample
48 % saccharose and 4 % magnesium sulfate (MgSO ·7H O).
4 2
equivalent to approximately 300 mg of trisodium citrate
The citrate lyase powder should be stored as specified by the
dihydrate in distilled water and dilute to 200 mL. Dilute a 3.0
supplier.
mL aliquot of this solution to 100 mL with water. This is the
6.4 Disodium b-Nicotinamide Adenine Dinucleotide, Re-
sample test solution.
duced Form Solution (0.0032 M)—Dissolve 10 mg of disodi-
8.2 During the following steps, use the appropriate micropi-
umb -nicotinamide adenine dinucleotide, reduced form, (b-
pet for the 10 and 100-µLvolumes, replacing the tip after each
NADH) in 4.0 mL of water. The b-NADH should be
addition. Standard volumetr
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