EN 18034:2024
(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 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.
Algen und algenbasierte Produkte oder Zwischenprodukte - Verfahren zur Probenentnahme und Analyse - Quantifizierung des Chlorophyll a Gehalts
Dieses Dokument legt ein Laborverfahren zur Quantifizierung von Chlorophyll a in Algen fest. Das Verfahren wurde ursprünglich an der Mikroalgenart Nannochloropsis sp. und einer wärmebehandelten Algenprodukt-Tomatensuppe mit Nannochloropsis sp.-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 sp. und der Makroalgenart Saccharina latissima untersucht. Die Mikroalgenarten Nannochloropsis sp. und Phaeodactylum sp. 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 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 l’espèce de microalgue Nannochloropsis et sur une soupe à la tomate à base de produit d’algue traité thermiquement supplémenté avec Nannochloropsis sp. et les macroalgues Ulva sp., Furcellaria lumbricalis et Saccharina latissima. Lors d’un essai interlaboratoires, la méthode a été soumise à essai sur la microalgue Nannochloropsis sp. et la macroalgue Saccharina latissima. Les espèces de microalgues Nannochloropsis et Phaeodactylum et de macroalgues Ulva ainsi que 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
Ta dokument določa laboratorijsko metodo za določevanje vsebnosti klorofila a v algah. Metoda je bila prvotno preskušena in ovrednotena z vrsto mikroalg Nannochloropsis sp. v toplotno obdelani paradižnikovi juhi iz alg z dodatkom Nannochloropsis sp. ter vrstami makroalg Ulva sp., Furcellaria lumbricalis in Saccharina latissima. Med medlaboratorijskim preskušanjem je bila metoda preskušena z vrsto mikroalg Nannochloropsis sp. in vrsto makroalg Saccharina latissima.
Vrsti mikroalg Nannochloropsis sp. in Phaodactlylum sp. ter vrsti makroalg Ulva sp. in Saccharina latissima so bile preskušene z medlaboratorijskim preskusom. Ta dokument je potrjen samo za klorofil a, vendar ga je mogoče uporabljati tudi za druge klorofile.
General Information
Standards Content (Sample)
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 (see 8.5.2).
6.2.1 C18 stationary phase, e.g. Nova-pak® 4 μm (3,9 × 150 mm).
For the double gradient (see 8.5.3).
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 1,5 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.
Nova-pak® is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
Zorbax Eclipse® is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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:
— 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 extraction procedure, except for the weighing, shall be performed under a fume hood.
The extraction procedure shall be conducted through the following steps:
1) weigh a test portion of approximately 25 mg of the test sample in a 10 mL pyrex screw capped tube
(i.e. weight test portion);
2) record the weight to the nearest 0,01 mg;
3) add approximately 850 mg glass beads;
4) add 5 mL methanol;
5) vortex for 30 s;
6) centrifuge at 450 g for 10 min;
7) transfer the solvent to a 20 mL pyrex screw capped tube, e.g. by using a Pasteur pipette;
8) leave the pellet in the 10 mL pyrex tube;
9) repeat step 4) to 8) three more times, which should result in a pooled extract of 4 consecutive
extractions;
10) homogenize the pooled extract with a total volume of 20 mL by turning the tube upside down;
11) transfer the extract to the HPLC vial with a syringe using a 0,20 μm filter.
The chlorophyll concentration decreases during the storage of extracts, even at temperatures of −20 °C
and −80 °C. Therefore, extracts should be put on HPLC as soon as possible after the extraction.
8.2 Determination of the concentration of the chlorophyll a standard
For the calibration curve, the concentration of the chlorophyll a standard shall be measured
spectrophotometrically as described below using published extinction coefficients found in Table 1.
Make a mother solution with a volume fraction of 90 % acetone, containing 0,025 mg/mL BHT, with a
concentration of the standard of 1 μg/mL.
Keep solutions in fume hood and far away from direct light. Absorbance (Amax) is measured in a 1-cm
cuvette at the standard's wavelength λmax (given with the published extinction coefficient) and at
750 nm to correct for light scattering. Note that the absorbance at known λmax of the chlorophylls should
be in the range of 0,2-0,8. If the absorbance is outside this range, then dilute the solution until the
absorbance is within this range.
Also for newly purchased chlorophyll standards the concentration should be measured
spectrophotometrically, because some degradation can already have taken place after the concentration
determination by the supplier.
In order to determine the concentration of chlorophylls, the following formula shall be used:
Ai λimax − Ai 750
( ) ( )
CiSTD= 10³
(1)
b α
where
CiSTD is the concentration (in μg/mL) of the standard for pigment i;
Ai(λimax) is the absorbance at λmax;
Ai(750) is the absorbance 750 nm;
b is the path length of the cuvette (cm) (= 1 cm); and
α -1 -1
is the weight-specific extinction coefficient (L g cm ) of chlorophyll i (see Table 1).
Table 1 — Extinction coefficients of chlorophylls and degradation products based on Appendix G
of Jeffrey et al. [1]
Solvent λmax (nm) -1 -1)
Name pigment α(L g cm
Chlorophyll a (MVChla) 90 % acetone 664,3 87,67
Chlorophyll b 90 % acetone 646,8 51,36
Chlorophyll c2 90 % acetone 443,8 374,00
Pheophorbide a 90 % acetone 667,0 74,20
Pheophytine a 90 % acetone 667,0 51,20
8.3 Preparation of the calibration curve
A calibration curve shall be prepared by diluting chlorophyll standards with the methanol extraction
solvent. Multipoint calibrations shall always be performed. Therefore, at least 6 dilutions of the mother
solution should be prepared, with a concentration range between 0,01 to 1,5 μg/mL. For every dilution,
the peak area shall be determined twice by HPLC, using the method as described in 8.5, and the calibration
curve shall be made as peak area versus concentration. A calibration curve shall be made based on the
duplicate results for each concentration. The resulting calibration curves should be linear according to:
y ax+ b (2)
where
y is the absorbance;
x is the concentration in μg/mL;
a is the slope;
b is the y-intercept.
It should be noted that the determination coefficients (R values) may never be less than 0,99.
A new calibration curve shall be constructed in the following cases:
• a new reference standard has been bought;
• a new type of chlorophyll has to be analysed;
• another gradient is used;
• the type of stationary phase used, is changed;
• the settings of the HPLC method have been changed;
• the HPLC has been moved to another location.
8.4 Quality assurance and quality control equipment
Calibration curves should be checked regularly. At least, once a year new chlorophyll standards shall be
purchased to make calibration curves.
When initiating a new sequence of samples on the HPLC device, 3 quality checks from the dilution series
should be performed in order to ensure the quality assurance and quality control of the equipment. For
these quality checks, 3 vials with known concentrations, which were previously run when creating
calibration curves, should be used. It does not matter which concentration is chosen, as long as the
concentration was initially measured when creating the standard curve, in order to enable the
comparison of the resulting peak areas with the earlier obtained ones. The quality is considered good,
when the deviation in the resulting concentrations is not over 10 %.
After measuring the chlorophyll standards for the first time, the cap on the vials shall be renewed. The
vials shall be stored at a temperature of minimum −20 °C. If possible, storage at −80 °C is recommended.
=
8.5 HPLC analysis
8.5.1 HPLC setup
It is recommended to use the triple gradient (see 8.5.2) with the C18 stationary phase (see 6.2.1). In case
your equipment is not suited for applying a triple gradient, the double gradient (see 8.5.3) may be used
with the C8 stationary phase (see 6.2.2).
8.5.2 Triple gradient
8.5.2.1 Mobile phases
• A: Mixture of methanol with a volume fraction of 80 % and 0,5M ammonium acetate buffer in purified
water, e.g. Milli-Q pH 7,2 with a volume fraction of 20 %.
• B: Mixture of acetonitrile with a volume fraction of 90 %, and purified water, e.g. Milli-Q with a volume
fraction of 10 %.
• C: Ethyl acetate.
8.5.2.2 Settings
Table 2 — Triple gradient settings
Time (min) A (%) B (%) C (%)
0 100 0 0
3 0 100 0
14 0 50 50
16 50 0 50
22,5 20 0 80
26 20 0 80
30 0 0 100
32 80 0 20
33 100 0 0
a) Injection volume: 40 μL;
b) Flow rate: 0,7 mL/min;
c) Column T: 30 °C;
d) Sample T: 10 °C;
e) PDA: 440 nm (for pheophytin the absorbance at 410 nm is used).
8.5.3 Double gradient
8.5.3.1 Mobile phase
• A: A mixture of methanol with a volume fraction of 70 % and 0,028M TBAA (tetrabutylammonium
acetate) in purified water, e.g. Milli-Q with a volume fraction of 30 %.
• B: methanol.
8.5.3.2 Settings
Table 3 — Double gradient settings
Time (min) A B
0 95 5
27 5 95
34 5 95
35 0 100
38 0 100
40 95 5
46 95 5
a) injection volume: 100 μL;
b) flow rate: 0,9 mL/min;
c) column T: 60 °C;
d) sample T: 4 °C;
PDA: 440 nm (for pheophytin a the absorbance at 410 nm is used).
8.5.4 Identification
In order to identify chlorophyll a, the retention time and the spectrum should be consistent with the
chlorophyll standard. A maximum deviation in retention time of 5 % is allowed.
During the integration and processing of the results, the peaks shall be integrated consistently. It may
occur that several peaks of chlorophylls are detected next to each other. When this is the case, the areas
of all peaks with the same spectrum as the chlorophyll standard shall be included. Annex C contains
several examples of chromatograms and spectra to illustrate this.
9 Calculation of chlorophyll a concentration
9.1 General
The chlorophyll a concentration shall be calculated from the calibration curves by:
Achlorophyll a− intersection
( )
Cchlorophyll a=
(3)
Vx
slope x
Mf
Where
C is the concentration of chlorophyll a in the sample (μg/g)
chlorophyll a
A is the peak area of chlorophyll a
chlorophyll a
intersection is the the value b from Formula (2)
slope is the the value a from Formula (2)
Vx is the extraction volume (in mL)
Mf is the weight of sample (in g)
9.2 Interlaboratory reproducibility
A maximum relative standard deviation of 10 % shall be obtained when analysing three test portions of
the same test sample.
During the statistical evaluation of the Interlaboratory Trial (ILT) and Round Robin (RR) for the
chlorophyll analysis method an acceptable maximal standard deviation of 25 % of the consensus value
was used.
Based on the results of the ILT and the RR (Annex B), the method has proven to provide acceptable results
for the several algae species. The 25 % acceptance criterium was not fulfilled for the macroalgae species
Ulva sp. Robust statistics could not be performed on the data obtained by the RR as a result of heavily
deviating results of three laboratories, of which after consultation no proper reason could be found
and/or no reaction was received of some of these laboratories.
Based on the results of the RR the RSD value is rather stable for the three other algae species. Therefore,
R
it is expected that the characteristics shown in Table 4 are also applicable to other algae species.
Table 4 — Quality parameters of the Round Robin results of chlorophyll a method
Algae Species Chlorophyll a 1 σ-value RSD R R
R abs rel
content (absolute
(in %) (in %) (in %)
(absolute mass mass
percentage) percentage)
Nannochloropsis sp. 2,8 0,73 26 2,0 73
Phaeodactylum sp. 0,67 0,13 19 0,4 54
Saccharina latissima 0,0061 0,0019 31 0,01 87
Key
RSD Relative standard deviation of the interlaboratory reproducibility
R
R Absolute Interlaboratory Reproducibility
abs
R Relative Interlaboratory Reproducibility
rel
10 Test Report
The test report shall specify the following information:
a) all information necessary for the complete identification of the sample;
b) the sampling method used, if known;
c) the test method used, with reference to this protocol, including the details of the stationary phase
and the gradient;
d) all operating details not specified in this protocol, or regarded as optional, together with details of
any incidents which may have influenced the test result(s);
e) the content of chlorophyll a, expressed in mg/g relative to dry weight;
f) the uncertainty associated to the test result(s);
g) the date of the test.
Annex A
(informative)
Chlorophyll standards and standard curves for calibration
For the identification and quantification, chlorophyll standards from DHI are recommended given their
high quality and purity.
Category and CAS numbers of chlorophylls and degradation product can be found in Table A.1.
Table A.1 — Chlorophylls and degradation product and their category and CAS number (N°)
Name Pigment Category N° CAS N°
Chlorophyll a C5753 479–61–8
Chlorophyll b PPS-CHLB 519–62–0
Chlorophyll c2 PPS-CHLC2 27736–03–4
Chlorophyll c3 PPS-CHLC3 111308–93–1
Pheophytin a PPS-PHAE 603–17–8
Annex B
(informative)
Results of Interlaboratory Trial and Round Robin for
chlorophyll a, b and c
B.1 Introduction
In addition to the results already described, an Interlaboratory Trial and Round Robin have been
conducted to optimize the standard. The main results are presented in this Annex.
The statistical approach can be found in the report on the Interlaboratory trial for algae and algae
products [2].
B.2 Results of the Interlaboratory Trial for chlorophyll a
B.2.1 Introduction
From April 2021 to June 2021 an interlaboratory trial (ILT) for chlorophyll in micro- and macroalgae was
organized by Wageningen Food Safety Research, part of Wageningen University and Research, on behalf
of CEN TC 454 on algae and algae products. This ILT enabled participants to test the analytical
performance according to the ‘Draft protocol for analysis of chlorophyll in algae and algae based
products’.
For this ILT, two materials were sent to the participants:
— Material A: Saccharina latissima, a brown macroalgae;
— Material B: Nannochloropsis sp., a microalgae species.
Ten laboratories registered for the analysis of chlorophyll, but most of them decided to withdraw their
participation after receipt of the draft protocol.
B.2.2 Material A - Saccharina latissima
Three quantitative results were reported and one participant did not detect the presence of chlorophyll
a. During homogeneity testing the mean of the 20 analyses was 0,05 % chlorophyll a. Figure B.1 shows
the results of the laboratories and the homogeneity test.
Figure B.1 — Graphical representation of the reported results for chlorophyll a in material A
B.2.3 Material B – Nannochloropsis sp
Four quantitative results were reported. During homogeneity testing the mean of the 20 analyses was
1,92 % chlorophyll a. Figure B.2 shows
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