ASTM D3598-89(2009)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D3598 − 89 (Reapproved2009)
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 formation of β-nicotinamide adenine dinucleotide, oxidized
form, is directly proportional to the concentration of citrate.
1.1 This test method covers the enzymatic determination of
citrate in both liquid and solid synthetic detergents. The test
4. Interferences
method is applicable to most detergents containing citrate at a
4.1 The test method is highly specific for citrate. Other
minimum concentration of approximately 5 % (1-8).
organic acids, for example, cisand trans-aconitic, d,l-isocitric,
1.2 The values stated in SI units are to be regarded as
α-ketoglutaric, oxalic, succinic, or tartaric acids, do not inter-
standard. No other units of measurement are included in this
fere.
standard.
4.2 Although low levels of zinc or magnesium, or both, are
1.3 This standard does not purport to address all of the
required as an activator for the enzyme citrate lyase, exces-
safety concerns, if any, associated with its use. It is the
sively high levels of divalent metallic ions including zinc and
responsibility of the user of this standard to establish appro-
magnesium will cause inactivation of the enzyme and poten-
priate safety and health practices and determine the applica-
tially interfere with the test method (7).
bility of regulatory limitations prior to use. Material Safety
4.3 The test method is not applicable to those detergents
Data Sheets are available for reagents and materials. Review
containing components with excessive absorptivity at 340 nm
them for hazards prior to usage.
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
5.1 Interval Timer.
Alkaline Detergents
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
volume of 4 mL.
products, oxaloacetate, is reduced to malate by malic dehydro-
genase (L-malate: NAD oxidoreductase; EC 1.1.1.37) with the
6. Reagents
simultaneous oxidation of reduced β-nicotinamide adenine
6.1 Purity of Reagents—Reagent grade chemicals shall be
dinucleotide to β-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,
where such specifications are available. Other grades may be
This test method is under the jurisdiction of ASTM Committee D12 on Soaps
used, provided it is first ascertained that the reagent is of
and Other Detergents and is the direct responsibility of Subcommittee D12.12 on
sufficiently high purity to permit its use without lessening the
Analysis and Specifications of Soaps, Synthetics, Detergents and their Components.
accuracy of the determination.
Current edition approved Oct. 1, 2009. Published December 2009. Originally
approved in 1977 as D3598 – 77 T. Last previous edition approved in 2003 as
D3598 – 89 (2003). DOI: 10.1520/D3598-89R09.
2 4
The boldface numbers in parentheses refer to the references at the end of this Reagent Chemicals, American Chemical Society Specifications, American
test method. Chemical Society, Washington, DC. For suggestions on the testing of reagents not
For referenced ASTM standards, visit the ASTM website, www.astm.org, or listed by the American Chemical Society, see Analar Standards for Laboratory
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Standards volume information, refer to the standard’s Document Summary page on and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
the ASTM website. MD.
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D3598 − 89 (2009)
6.2 Purity of Water—Unless otherwise indicated, references S 3 V
C 5 (1)
I,II
to water shall be understood to mean Type II reagent water 10
conforming to Specification D1193.
where:
6.3 Citrate Lyase Solution (40 units/mL)—Add sufficient
C = concentration of trisodium citrate dihydrate in the
I,II
cold water to a vial of citrate lyase containing a premeasured
standard Solutions I or II,µ g/mL,
weight of enzyme protein such that the resulting solution will
S = standard weight of TSC, mg, and
contain 40 units/mL; for example, 2.0 mL of water is added to
V = volume taken for the final dilution, mL.
a vial containing 5 mg of enzyme protein with an activity of 16
Prepare all solutions fresh daily.
units/mg of enzyme protein. One unit of activity will convert
6.10 Zinc Chloride Solution (0.003 M)—Dissolve 41 mg of
1.0 µmol of citrate to oxaloacetate per minute at pH 7.6 at
zinc chloride in 100 mL of water.
25°C. The citrate lyase solution should be maintained in an ice
bath for the duration of the analyses and can be used for 5 days
7. Sampling
if refrigerated. Citrate lyase (EC 4.1.3.6) from Aerobacter
7.1 Collect the sample in accordance with Test Methods
aerogenes is commercially available as a lyophilized powder
D501.
containing approximately 24 % citrate lyase, 24 % albumin,
48 % saccharose and 4 % magnesium sulfate (MgSO ·7H O).
4 2
8. Procedure
The citrate lyase powder should be stored as specified by the
8.1 Dissolve an accurately weighed detergent sample
supplier.
equivalent to approximately 300 mg of trisodium citrate
6.4 Disodium β-Nicotinamide Adenine Dinucleotide, Re-
dihydrate in distilled water and dilute to 200 mL. Dilute a 3.0
duced Form Solution (0.0032 M)—Dissolve 10 mg of
mL aliquot of this solution to 100 mL with water. This is the
disodiumβ -nicotinamide adenine dinucleotide, reduced form,
sample test solution.
(β-NADH) in 4.0 mL of water. The β-NADH should be
8.2 D
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