ISO 21264:2019

INTERNATIONAL ISO
STANDARD 21264
First edition
2019-07
Surface active agents — Detergents
— Determination of alkylphenol
ethoxylates
Agents de surface — Détergents — Dosage des alkylphénols éthoxylés
Reference number
©
ISO 2019
© ISO 2019
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ii © ISO 2019 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 1
6 Apparatus . 2
7 Procedure. 2
7.1 Standard preparation . 2
7.2 Sample preparation . 2
7.3 Analysis. 3
7.3.1 Chromatographic condition . 3
7.3.2 Qualitative determination . 3
7.3.3 Calibration curve . 3
7.3.4 Quantitative determination . 4
8 Expression of results . 4
8.1 Calculation . 4
8.2 E valuation . 4
8.3 Precision . 5
8.3.1 Repeatability . 5
8.3.2 Reproducibility limit. 5
9 Test report . 5
Annex A (informative) Chromatogram of alkylphenol ethoxylates (APEOs) .6
Annex B (informative) Identification test . 7
Annex C (informative) Statistical data from the ring carried out . 9
Foreword
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This document was prepared by Technical Committee ISO/TC 91, Surface active agents.
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iv © ISO 2019 – All rights reserved

INTERNATIONAL STANDARD ISO 21264:2019(E)
Surface active agents — Detergents — Determination of
alkylphenol ethoxylates
1 Scope
This document provides a method for the determination of alkylphenol ethoxylates (APEOs) in
surfactants using high performance liquid chromatography (HPLC) and detected with diode array
detector (DAD) or fluorescence detector (FLD).
This method is appropriate for the detection and quantification of APEOs in surfactants.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 607, Surface active agents and detergents — Methods of sample division
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
4 Principle
The sample is weighed into a vial and extracted in methanol using an ultrasonic bath. Subsequently
the extract is filtered and analysed using high performance chromatography system with diode array
detector (DAD) or fluorescence detector (FLD). Quantification is achieved by external quantification
method and using the relationship chromatographic peak through its unique its retention time.
5 Reagents and materials
During the analysis, use only reagents of recognized analytical grade and the water of quality for HPLC
analysis.
5.1 Methanol, HPLC Grade.
1)
5.2 Octylphenol ethoxylates (OPEO, 2 ≤ n ≤ 12), for example, Triton X-100 , CAS no. 9002-93-1,
n
Sigma-Aldrich Part Number T9284.
1) Triton X-1000 ®, CAS no. 9002-93-1, Sigma Aldrich Part Number T9284 is an example of products. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO
of the product named. Equivalent products may be used if they can be shown to lead to the same results.
2)
5.3 Nonylphenol ethoxylates (NPEO, 3 ≤ n ≤ 18), for example, IGEPAL CO-630 , CAS no. 9016-45-
n
9, Sigma-Aldrich Part Number 542334.
NOTE There is a possibility that reagents of the grade of OPEO and NPEO are not available.
6 Apparatus
Normal laboratory apparatus and, in particular, the following:
6.1 Analytical balance, weighing to an accuracy of 0,1 mg.
6.2 Ultrasonic bath, 40 kHz, with thermostat.
6.3 Membrane filter, polyamide, 0,45 μm.
6.4 Volumetric flasks, 10 ml, 50 and 100 ml capacity.
6.5 High-performance Liquid Chromatography (HPLC) with DAD or FLD detector.
6.6 Reverse phase column with guard column, a stainless-steel column 150 mm and 2,1 mm in
internal diameter filled with 3,5 µm ODS (C ) as stationary phases.
7 Procedure
7.1 Standard preparation
7.1.1 Prepare 1 000 mg/l stock solutions of each APEOs from standard materials or certified solutions in
methanol. 100 mg of the OP EO (5.2) and NP EO (5.3) are dissolved in different 100 ml volumetric flasks
n n
with methanol and filled up to the mark respectively.
Depending on the stock concentrations prepared, the solubility at that concentration will have to be
ensured.
7.1.2 Prepare calibration curve for samples of each APEO stock standard solution at required
concentrations by adding aliquots of stock solutions to a 100 ml volumetric flask and then diluted with
methanol to the desired calibration level. If lower reporting limit is required, lower concentration of
calibration standard shall be prepared to cover the reporting limit. Each calibration curve is prepared
using the respective stock solution. The preparation of the Level 5 standard can be accomplished using
different volumes and concentrations of stock solutions as is accustomed in the individual laboratory.
The calibration vials shall be used within 24 h to ensure optimum results. Stock calibration standards
are routinely replaced every 14 days if not previously discarded for quality control failure.
7.2 Sample preparation
The laboratory samples shall be prepared and stored in accordance with ISO 607.
The test samples can be in liquid, paste or solid form. Liquefaction and homogenization can be necessary
for paste and solid substances. Liquefaction can be accelerated by gentle warming (below 50 °C).
2) IGEPAL CO-630 ®, CAS no. 9016-45-9, Sigma-Aldrich Part Number 542334 is an example of products. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO
of the product named. Equivalent products may be used if they can be shown to lead to the same results.
2 © ISO 2019 – All rights reserved

Weigh approximately 1,0 g (accurate to 0,1 mg) of the test sample to 50 ml volumetric flasks and make
up to volume with methanol. Put the flask into an ultrasonic water bath and extract at least 30 min.
Then filter the extracts through a 0,45 μm nylon filter disc for analysis.
7.3 Analysis
7.3.1 Chromatographic condition
As the HPLC equipment system of the laboratories can vary, no general valid instructions can be
provided for the chromatographic analysis. The following parameters are provided as an example.
—  Column: StableBond SB-C18, 150 mm × 2,1 mm (i.d.) or equivalent
—  Stationary phase: C18 reverse phase, 3,5 µm
—  Eluent 1: Methanol
—  Eluent 2: Water
—  Gradient elution: see Table 1
—  Flow rate: 0,3 ml/min
—  Column temperature: 40 °C
—  Injection: 10,0 μl
—  Detection: FLD or DAD, spectrograph
—  Quantification: for DAD at 225 nm
for FLD with Ex = 230 nm and Em = 296 nm
Table 1 — Gradient elution for HPLC
Time Eluent 1 Eluent 2
min % %
0,00 85 15
20,00 98 2
25,00 98 2
26,00 85 15
30,00 85 15
7.3.2 Qualitative determination
Injection volumes of all calibration standards and extraction solutions at concentration gradient are
made at 10,0 μl volume by autosampler under selected conditions. An aliquot of the extraction solution
is automatically ejected onto a reverse phase column (6.6) for sample component separation. Under the
chromatographic conditions given, the detection and identification of the ch
...