SIST EN 18034:2025

SLOVENSKI STANDARD
01-februar-2025
Alge in izdelki iz alg - Metode vzorčenja in analize - Določevanje vsebnosti klorofila
Algae and algae products - Methods of sampling and analysis - Determination of
chlorophyll a content
Algen und algenbasierte Produkte oder Zwischenprodukte - Verfahren zur
Probenentnahme und Analyse - Quantifizierung von Chlorophyll
Algues et produits ou intermédiaires à base d'algues - Méthodes d'échantillonnage et
d'analyse - Détermination de la teneur en chlorophylle a
Ta slovenski standard je istoveten z: EN 18034:2024
ICS:
13.020.55 Biološki izdelki Biobased products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 18034
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2024
EUROPÄISCHE NORM
ICS 13.020.55
English Version
Algae and algae products - Methods of sampling and
analysis - Determination of chlorophyll a content
Algues et produits ou intermédiaires à base d'algues - Algen und algenbasierte Produkte oder
Méthodes d'échantillonnage et d'analyse - Zwischenprodukte - Verfahren zur Probenentnahme
Détermination de la teneur en chlorophylle a und Analyse - Quantifizierung des Chlorophyll a
Gehalts
This European Standard was approved by CEN on 4 November 2024.

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. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 18034:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Apparatus . 6
6 Reagents and materials . 6
7 Sampling and preparation of the sample . 8
8 Procedure . 8
9 Calculation of chlorophyll a concentration . 12
10 Test Report . 13
Annex A (informative) Chlorophyll standards and standard curves for calibration . 15
Annex B (informative) Results of Interlaboratory Trial and Round Robin for chlorophyll a, b
and c . 16
B.1 Introduction . 16
B.2 Results of the Interlaboratory Trial for chlorophyll a . 16
B.3 Results of the Interlaboratory Trial for chlorophyll b and c . 18
B.4 Results of the Round Robin for chlorophyll a . 19
B.5 Results of the Round Robin for chlorophyll b . 26
B.6 Results of the Round Robin for chlorophyll c . 28
B.7 Conclusions of the Round Robin . 31
Annex C (informative) Identification of chlorophyll a . 32
C.1 Identification of chlorophyll a . 32
C.2 Chlorophyll a standard: Triple gradient – C18 stationary phase . 32
C.3 Chlorophyll a standard: Double gradient – C8 stationary phase . 35
Bibliography. 39

European foreword
This document (EN 18034:2024) has been prepared by Technical Committee CEN/TC 454 “Algae and
algae products”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by June 2025, and conflicting national standards shall be
withdrawn at the latest by June 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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 the United
Kingdom.
Introduction
This document has been prepared by the experts of CEN/TC 454 'Algae and algae products'.
The European Committee for Standardization (CEN) was requested by the European Commission (EC) to
draft European standards or European standardization deliverables to support the implementation of
Article 3 of Directive 2009/28/EC for algae and algae-based products or intermediates.
This request, presented as Mandate M/547 , also contributes to the Communication on “Innovating for
Sustainable Growth: A Bio economy for Europe”.
The former working group CEN Technical Board Working Group 218 “Algae”, was created in 2016 to
develop a work programme as part of this Mandate. The technical committee CEN/TC 454 'Algae and
algae products' was established to carry out the work programme that will prepare a series of standards.
The interest in algae and algae-based products or intermediates has increased significantly in Europe as
a valuable source including but not limited to, carbohydrates, proteins, lipids, and several pigments.
These materials are suitable for use in a wide range of applications from food and feed purposes to other
sectors, such as textile, cosmetics, biopolymers, biofuel and fertilizer/biostimulants. Standardization was
identified as having an important role in order to promote the use of algae and algae products.
The work of CEN/TC 454 should improve the reliability of the supply chain, thereby improving the
confidence of industry and consumers in algae, which include macroalgae, microalgae, cyanobacteria,
Labyrinthulomycetes, algae-based products or intermediates and will promote and support
commercialisation of the European algae industry.
Given the importance of chlorophyll a in algae and algae products, it is important to have a good
standardized method to determine the amount of chlorophyll a.

Available at https://ec.europa.eu/growth/tools-databases/mandates/index.cfm?fuseaction=refSearch.search#
1 Scope
This document specifies a laboratory method for the determination of chlorophyll a content in algae. The
method was initially tested and evaluated on the microalgae species Nannochloropsis sp. and a heat
treated algal product tomato soup with Nannochloropsis sp. supplement, and the macro algae species Ulva
sp., Furcellaria lumbricalis, and Saccharina latissima. During an Interlaboratory Trial the method was
tested on the microalgae species Nannochloropsis sp. and the macro algae species Saccharina latissima.
The microalgae species Nannochloropsis sp. and Phaodactlylum sp. and the macro algae species Ulva sp.
and Saccharina latissima were tested in a Round Robin test. This document is only validated for
chlorophyll a, but it can be used for other chlorophylls as well.
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 17399, Algae and algae products — Vocabulary
EN 17605:2022, Algae and algae products — Methods of sampling and analysis — Sample treatment
3 Terms and definitions
For the purpose of this document, the terms and definitions given in EN 17399 and the following 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/
3.1
chlorophyll
chlorophyll a, b, c1, c2 and d excluding degradation products
3.2
carotenoid
accessory pigment of the class of tetraterpenoids including carotenes and xanthophylls
3.3
carotene
unoxygenated (oxygen free) carotenoid
EXAMPLE α-carotene, β-carotene, and lycopene.
3.4
chlorophyll standard
solution with defined concentration of the chlorophyll of interest that is used to determine the analyte of
interest with the help of standard curves
Note 1 to entry: This is explained in further detail in Annex A.
3.5
degradation product
result of physical, biological or chemical interaction by e.g. enzymatic, heat or acid treatment, leaving
behind a fully or partial degraded compound
3.6
mother solution
initial part of the standard solution, from which the dilutions for making standard curves are taken
3.7
pheophytin a
degradation product of chlorophyll a
3.8
pigment
colouring matter in the cells and tissues of plants
3.9
xanthophylls
carotenoid molecule containing oxygen
EXAMPLE Lutein, zeaxanthin.
4 Principle
The chlorophylls are extracted from the sample with methanol and separated using HPLC (High
Performance Liquid Chromatograph). The separated components are analysed at 440 nm using a
photodiode array detector.
5 Apparatus
Usual laboratory apparatus and, in particular, the following:
5.1 Analytical balance, with an accuracy of preferably 0,01 mg.
5.2 High Performance Liquid Chromatograph (HPLC) with a Photodiode Array detector (PDA).
5.3 Centrifuge.
5.4 Vortex.
5.5 Spectrophotometer.
5.6 Fume hood.
6 Reagents and materials
6.1 HPLC reagents
All reagents should be HPLC grade.
For the triple gradient (see 8.5.2).
6.1.1 Mixture of methanol with a volume fraction of 80 % and 0,5M ammonium acetate buffer in
purified water, e.g. Milli-Q, with a volume fraction of 20 %.
6.1.2 Mixture of acetonitrile with a volume fraction of 90 %, and purified water, e.g. Milli-Q with
a volume fraction of 10 %.
6.1.3 Ethyl acetate.
For the double gradient (see 8.5.3).
6.1.4 Mixture of methanol with a volume fraction of 70 %, and 0,028M TBAA
(tetrabutylammonium acetate) in purified water, e.g. Milli-Q.
6.1.5 Methanol.
6.2 HPLC column
For the triple gradient (s
...