FprEN 18034
(Main)Algae and algae products - Methods of sampling and analysis - Determination of chlorophyll a content
Algae and algae products - Methods of sampling and analysis - Determination of chlorophyll a content
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 and a heat treated algal product tomato soup with Nannochloropsis 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 and the macro algae species Saccharina latissima. The microalgae species Nannochloropsis and Phaodactlylum 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.
If possible, an attempt will be made to incorporate an alternative method than the HPLC method proposed into the standard (informative or normative), on the condition that research on the use of a cheaper method fits into the budget allocated for this topic. The more, it is imperative that this additional method should obtain comparable results and accuracy as the HPLC method.
The schedules of all the additional experiments mentioned are based on the schedule of the standard on chlorophyll.
Algen und algenbasierte Produkte oder Zwischenprodukte - Verfahren zur Probenentnahme und Analyse - Quantifizierung von Chlorophyll
Dieses Dokument legt ein Laborverfahren zur Quantifizierung von Chlorophyll a in Algen fest. Das Verfahren wurde ursprünglich an der Mikroalgenart Nannochloropsis und einer wärmebehandelten Algenprodukt-Tomatensuppe mit Nannochloropsis-Zusatz und an den Makroalgenarten Ulva sp, Furcellaria lumbricalis und Saccharina latissima geprüft und evaluiert. Während eines Ringversuchs wurde das Verfahren an der Mikroalgenart Nannochloropsis und der Makroalgenart Saccharina latissima untersucht. Die Mikroalgenarten Nannochloropsis und Phaeodactylum und die Makroalgenarten Ulva sp und Saccharina latissima wurden in einem Ringvergleich untersucht. Dieses Dokument ist nur für Chlorophyll a validiert, aber es kann auch für andere Chlorophyllarten verwendet werden.
Algues et produits ou intermédiaires à base d'algues - Méthodes d'échantillonnage et d'analyse - Détermination de la teneur en chlorophylle a
Le présent document spécifie une méthode de détermination en laboratoire de la teneur en chlorophylle a des algues. La méthode a été initialement soumise à essai et évaluée sur la microalgue Nannochloropsis et sur une soupe à la tomate à base de produit d’algue traité thermiquement contenant le complément Nannochloropsis et les macroalgues Ulva, Furcellaria lumbricalis et Saccharina latissima. Lors d’un essai interlaboratoires, la méthode a été soumise à essai sur la microalgue Nannochloropsis et la macroalgue Saccharina latissima. Les microalgues Nannochloropsis et Phaeodactylum et les macroalgues Ulva et Saccharina latissima ont été évaluées au cours d’un essai comparatif. Le présent document n’est validé que pour la chlorophylle a, mais il peut également être utilisé pour d’autres chlorophylles.
Alge in izdelki iz alg - Metode vzorčenja in analize - Določevanje vsebnosti klorofila
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-januar-2024
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: prEN 18034
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
prEN 18034
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2023
ICS
English Version
Algae and algae products or intermediates - Methods of
sampling and analysis - Determination of chlorophyll a
content
Algues et produits à base d'algues - Méthodes Algen und Algenprodukte - Methoden zur
d'échantillonnage et d'analyse - Détermination de la Probeentnahme und Analyse - Bestimmung von
teneur en chlorophylle a Chlorophyll a gehalts
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 NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18034:2023 E
worldwide for CEN national Members.
prEN 18034:2023 (E)
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 . 7
8 Procedure . 8
9 Calculation of chlorophyll a concentration . 12
10 Test Report . 13
Annex A (informative) Chlorophyll standards and standard curves for calibration . 14
Annex B (informative) . 15
Annex C . 31
Bibliography. 38
prEN 18034:2023 (E)
European foreword
This document (prEN 18034:2023) 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.
This document has been prepared under a standardization request addressed to CEN by the European
Commission.
prEN 18034:2023 (E)
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.
Text in grey is fixed (based on CEN/TC 454 Decision 5-2019 taken by CEN/TC 454 on 2019-01-29 /
Subject: CEN/TC 454 – Approval of the General Introduction Text).
Available at http://ec.europa.eu/growth/tools-databases/mandates/index.cfm?fuseaction=refSearch.search#
prEN 18034:2023 (E)
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 and a heat treated
algal product tomato soup with Nannochloropsis 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 and the macro algae species Saccharina latissima. The
microalgae species Nannochloropsis and Phaodactlylum 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 - Terms and definitions
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.
3.1
chlorophylls
chlorophyll a, b, c1, c2 and d excluding degradation products
3.2
carotenoids
all accessory pigments of the class of tetraterpenoids including carotenes and xanthophylls
3.3
carotenes
unoxygenated (oxygen free) carotenoids such as 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 or chemical interaction by e.g. light, enzymatic, heat or acid treatment, leaving behind
a fully or partially degraded pigment compound
3.6
mother solution
initial part of the standard solution, from which the dilutions for making standard curves are taken
prEN 18034:2023 (E)
3.7
pheophytin a
degradation product of chlorophyll a
3.8
pigments
all coloured substances in algae and algae products, such as chlorophylls, carotenoids and
phycobiliproteins including their degradation products
3.9
xanthophylls
carotenoid molecules containing oxygen, such as lutein and zeaxanthin
4 Principle
The chlorophylls are extracted from the sample with methanol and separated using HPLC. 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.
5.7 Storage container, dark for storing the samples prior to analysis.
6 Reagents and materials
6.1 HPLC reagents.
All reagents should be HPLC grade.
For the triple gradient (see 8.6.1.1).
6.1.1 Mixture of methanol with a volume fraction of 80 % and 0,5M ammonium acetate buffer
pH 7,2 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 8.6.1.2):
6.1.4 Mixture of methanol with a volume fraction of 70 %, and 0,028 M TBAA
(tetrabutylammonium acetate) in purified water, e.g. Milli-Q.
prEN 18034:2023 (E)
6.1.5 Methanol.
6.2 HPLC column.
For the triple gradient (see 8.6.1.1).
6.2.1 C18 stationary phase, e.g. Nova-pak 4 μm (3,9 × 150 mm).
For the double gradient (see 8.6.1.2).
6.2.2 C8 stationary phase e.g. Zorbax Eclipse XDB-C8 3,5 μm (4,6 × 150 mm).
6.3 Pyrex screw capped tubes, 10 mL (1 / test portion) and 20 mL (1 / test portion).
6.4 Micropipettes.
6.5 Pasteur pipettes.
6.6 Glass beads, ∅ 0,75 mm to 1 mm.
6.7 HPLC vials and caps.
6.8 Syringe, with volume of 1 mL and filters (0,20 μm, ∅ 15 mm).
6.9 Methanol with a volume fraction of ⩾ 99,0 %.
6.10 Quartz cuvettes.
6.11 Chlorophyll standards, for the identification and quantification of chlorophyll, that shall comply
to the following quality parameters:
A concentration range from 0,5 to 3,0 μg/ml;
Dissolved in solvent
Transported on dry ice by the supplier
Stored after arrival at least at −20 °C and preferably at −80 °C
Recommendations for chlorophyll standards are given in Annex A.
6.12 Aluminium foil.
7 Sampling and preparation of the sample
7.1 Sampling
It is important that the laboratory receives a sample that has not been damaged or changed during
transport or storage.
Sampling is not part of the method specified in this document.
7.2 Sample treatment
Sampling and sample treatment are not parts of the method specified in this document. A recommended
sample handling procedure is given in EN 17605:2022 with the following adaptations / additions:
prEN 18034:2023 (E)
— fine grinding shall be conducted as defined in 3.13 in EN 17605:2022;
— transport shall be performed on dry ice;
— storage shall be at −80°C. Also, macroalgae need to be freeze-dried.
Extensive storage times of the test sample should be avoided. In case this could not be avoided, the test
sample should be freeze-dried again before usage.
Three test portions from the test sample shall be analysed as to be able to calculate the mean and standard
deviation.
The samples shall be protected from light at any time before and during the analysis, by using e.g.
aluminium foil.
Samples shall not be stored for over one month, because storage can degrade chlorophylls, even when
the samples are frozen.
8 Procedure
8.1 Extraction
The whole extr
...
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