prEN 18207
(Main)Algae and algae products – Determination of the Uronic Acids Content of Brown Seaweed and Alginate Products
Algae and algae products – Determination of the Uronic Acids Content of Brown Seaweed and Alginate Products
This document specifies a method for the quantitative determination of total
uronic acids by High-Performance Anion Exchange Chromatography coupled with
Pulsed Amperometric Detection (HPAEC-PAD) after acid hydrolysis of the
samples. It provides a single analysis method for determining mannuronic,
glucuronic, and guluronic acids in brown seaweed and alginate products.
Algen und Algenprodukte –-Bestimmung des Uronsäuregehalts von braunem Seetang und Alginatprodukten
Dieses Dokument legt ein Verfahren für die quantitative Bestimmung der gesamten Uronsäuren durch eine mit gepulster amperometrischer Detektion gekoppelte Hochleistungs-Anionenaustausch-Chromatographie (en: high performance anion exchange chromatography coupled with pulsed amperometric detection, HPAEC-PAD) nach einer Säurehydrolyse der Proben für Algen und Algenprodukte fest. Es legt ein Verfahren für die Bestimmung der folgenden Uronsäuren in einer einzigen Analyse fest: Mannuron-, Glucuron- und Guluronsäure in braunem Seetang und Alginatprodukten.
Algues et produits d’algues - Détermination de la teneur en acides uroniques des algues brunes et des produits à base d’alginate
Le présent document spécifie une méthode de détermination quantitative des acides uroniques totaux par chromatographie d’échange d’anions haute performance couplée à la détection par ampérométrie pulsée (HPAEC-PAD) après hydrolyse acide des échantillons d’algues et de produits d’algues. Il spécifie une méthode pour la détermination, en une seule analyse, des acides uroniques suivants : acides mannuronique, glucuronique et guluronique dans les algues brunes et les produits à base d’alginate.
Alge in izdelki iz alg - Določanje vsebnosti uronskih kislin v rjavih morskih algah in alginatnih izdelkih
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-september-2025
Alge in izdelki iz alg - Določanje vsebnosti uronskih kislin v rjavih morskih algah in
alginatnih izdelkih
Algae and algae products - Determination of the Uronic Acids Content of Brown
Seaweed and Alginate Products
Algen und Algenprodukte - Bestimmung des Uronsäuregehalts von braunem Seetang
und Alginatprodukten
Algues et produits d’algues - Détermination de la teneur en acides uroniques des algues
brunes et des produits à base d’alginate
Ta slovenski standard je istoveten z: prEN 18207
ICS:
13.020.55 Biološki izdelki Biobased products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
DRAFT
EUROPEAN STANDARD
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2025
ICS
English Version
Algae and algae products - Determination of the Uronic
Acids Content of Brown Seaweed and Alginate Products
Algues et produits à base d'algues - Détermination de Algen und Algenprodukte - Bestimmung des
la teneur en acide uronique de la biomasse de Polysaccharidgehalts von Makroalgenbiomasse
macroalgues
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 454.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18207:2025 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 4
5 Reagents . 4
6 Reagents and standard preparation . 5
7 Apparatus . 6
8 Sample analysis . 7
9 Calculation and expression results . 9
10 Precision . 10
11 Test report . 10
Annex A (informative) Results of Interlaboratory study for Uronic acids . 11
Annex B (informative) Chromatogram examples . 14
Annex C (informative) Trueness data . 16
Bibliography . 17
European foreword
This document (prEN 18207:2025) has been prepared by Technical Committee CEN/TC 454 “Algae and
algae products”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
1 Scope
This document specifies a method for the quantitative determination of total uronic acids by High
Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-
PAD) after acid hydrolysis of the samples for algae and algae products. It specifies a method for the
determination, in one single analysis of the following uronic acids: mannuronic, glucuronic and guluronic
acids in brown seaweed and alginate products.
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.
EN 17605:2022, Algae and algae products - Methods of sampling and analysis - Sample treatment
EN 17399:2024, Algae and algae products - Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions in EN 17399:2024 apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp/
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
Polysaccharides are hydrolysed under acidic conditions at 50°C. Hydrolysis duration depends on the
matrix to analyse (see Table 2). Hydrolysates are then diluted according to the sample type before
injection to the chromatographic system.
5 Reagents
5.1 General
Before using chemicals, refer to the safety data sheets and ensure that the safety precautions are applied.
5.2 Trifluoroacetic acid
CAS 76-05-1, HPLC grade
5.3 Formic acid
CAS 64-18-6, HPLC grade
5.4 Sulfuric acid 18 M
CAS 7664-93-9, HPLC grade
5.5 Sodium Tetraborate decahydrate
CAS 1303-96-4
5.6 Sodium hydroxide solution 50 %
CAS 1310-73-2, HPLC grade
5.7 Sodium acetate trihydrate
CAS 6131-90-4
5.8 Ultra-pure water
with a resistivity of 18,2 MΩ-cm
5.9 Mannuronic acid standard
CAS 6906-37-2
5.10 Guluronic acid standard
CAS 1986-15-8
5.11 Glucuronic acid standard
CAS 6556-12-3
6 Reagents and standard preparation
6.1 Primary hydrolysis solution (labelled SH)
Mix 2 volumes of ultra-pure water to 3 volumes of formic acid and thereafter add 1 volume of TFA (e.g.
20 ml of water + 30 ml of formic acid + 10 ml of TFA). Store solutions at room temperature until use.
6.2 Secondary hydrolysis solution (labelled SA1)
Dissolve 2,845 ± 0,005 g of sodium tetraborate decahydrate to 100 ml with HPLC sulfuric acid 18M. Mix
overnight for complete dissolution.
6.3 Uronic acid standards and calibration curve preparation
Prepare a 1 mg/ml stock solution for each uronic acid separately by weighing 10 mg of standard into a
10 ml volumetric flask and make up to the volume with water. Aliquot and store at - 20°C until use.
Prepare, in a volumetric flask, a solution containing each uronic acid at 100 µg/ml by adding 100 μl of
each 1 mg/ml stock solution and adjusting the volume to 1 ml with ultra-pure water.
Prepare the calibration samples using the following table.
Table 1 — Uronic acids samples for calibration curve
Volume of the 100 μg/ml Volume
Label
solution Of ultra-pure water
Mix - 5 μg/ml 25 475
Mix - 10 μg/ml 50 450
Mix - 25 μg/ml 125 375
Mix - 50 μg/ml 250 250
Mix - 75 μg/ml 375 125
Mix - 100 μg/ml 500 0
6.4 Chromatography solvents (mobile phases)
6.4.1 Sodium hydroxide (NaOH) solution at 200mM, HPLC grade
Use 10,5 ml of NaOH 50 % solution for one litre.
6.4.2 Sodium acetate solution at 250mM
In a volumetric flask, dissolve 34 g of sodium acetate trihydrate (or alternatively 20,5 g of anhydrous
sodium acetate) and make the volume to 1L with ultra pure water.
7 Apparatus
7.1 IC System
Ion chromatography system equipped with a quaternary pump and a pulsed amperometric detector.
Helium gas should also be available to pressurize and degas the eluents.
7.2 Chromatography column
CarboPac™ SA10,4 X 250 mm and the corresponding guard column.
7.3 Adjustable micropipettes
Volume 200 μl, and 1 000 μl and tips.
7.4 Vortex mixer
7.5 Analytical balance
Able to accurately measure 10 mg with a precision of 0,1 mg.
7.6 Thermomixer block
Able to maintain a temperature of 50 °C ± 2 °C and stirring speed up to 1000 rpm.
7.7 Screw cap microtubes
Volume 2 ml.
7.8 Vial with screw cap
Adapted to the autosampler used.
7.9 Centrifuge
10 000 rpm for microtubes.
8 Sample analysis
8.1 Sample preparation
Samples shall be dried and ground at 500 μm particle size maximum according to protocol described in
EN 17605:2022 ‘Algae and algae products - Methods of sampling and analysis - Sample treatment’.
Weigh 20 mg ± 0,5 mg of sample into a 2 ml screw cap microtube (note the exact weight).
8.2 Hydrolysis
For each sample matrix (brown seaweed and purified alginate), hydrolysis conditions are adapted and
summarized in Table 2.
— Weigh the sample mass as mentioned in Table 2 according to the sample (note the exact mass m1);
— Add SH solution (volume according to sample type);
— Add slowly SA1 solution (volume according to sample type).
WARNING — Addition of SA1 leads to a strong exothermic reaction.
— Pierce the cap of the tube to avoid overpressure;
— Incubate at 50 °C in a heating block during the time indicated in Table 1 according to the sample;
— At the end of the incubation, remove tubes for the heating block;
— Dilute 10 times (D10) to stop the reaction (100 µl in 900 µl of ultra-pure water);
— Centrifuge at 12 000 rpm during 10 min.;
— Dilute the sample before injection (see Table 2 for classical total dilution, including the D10
previously done).
Table 2 — Hydrolysis conditions
Alginates Brown seaweed
Sample weight (mg) 20 20
SH volume (µl) 600 600
SA1 volume (µl) 400 400
Hydrolysis time 1h30 2h30
Temperature 50 °C 50 °C
a
Classical total dilution 200 100
a
Classical total dilution: might be adapted according to samples
8.3 HPAEC separation
Allow the chromatographic system to stabilize before injecting standards and samples.
Make sure the system pressure and initial conditions are stable before performing injections.
— Before starting a series of analyses, inject two blanks (water) to condition the column;
— Perform HPLC under the conditions given below and in Table 3.
— Column temperature: 25 °C;
— Injection Volume: 10 µl;
— Flow rate: 1 ml per minute;
— Mobile phase A: ultra-pure water;
— Mobile phase B: sodium hydroxide 200 mM.
— Mobile phase C: sodium acetate 250 mM
— Detection: pulsed amperometry with gold electrode using the waveform described in Tabel 4.
Table 3 — Gradient steps for uronic acids separation
Time % A % B % C Curve
0.00 62,5 17,5 20,0 5
2.00 62,5 17,5 20,0 5
12.00 40,0 10,0 50,0 5
15.00 2,5 17,5 80,0 5
16.00 2,5 17,5 80,0 5
18.00 62,5 17,5 20,0 5
30.00 62,5 17,5 20,0 5
NOTE Operating conditions can vary depending on the apparatus.
Follow the supplier’s instructions.
Table 4 — Waveform for Pulsed Ameperometry Detection
Time Voltage Gain region
s V
0.00 +0,10 Off
0.20 +0,10 On
0.40 +0,10 Off
0.41 −2,00 Off
0.42 −2,00 Off
Time Voltage Gain region
s V
0.43 +0,60 Off
0.44 −0,10 Off
0.50 −0,10 Off
8.4 Peak identification and integration
Identify the uronic acids peaks in the sample solution by comparison with the retention times of the
corresponding peaks obtained in the calibration standards (Annex B). If a peak has not been integrated
correctly, call the recorded data and reintegrate.
To verify system stability, inject a mid-level standard a minimum of three times (5 × for USP
requirements) and ensure response and retention times have an RSD % < 2.
Check peaks are separated with a good resolution (baseline separation). If this is not the case, adapt the
chromatographic conditions (gradient, temperature, tubing length, etc.) accordingly.
9 Calculation and expression results
9.1 Calibration curve
Establish the calibration curve from the six different calibration standards for each uronic acid by plotting
the response (peak area of analyte against analyte concentration). Force the linear regression through
zero. Check the linearity of the calibration (the correlation coefficient R shall be above 0,99).
9.2 Uronic acid calculation
Results are expressed in gr
...
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