Appendices
C.3 Chlorophyll content (chl a, chl b, chl dev) of beech leaves in the early (May to June), mid (July to 744
August), and late (September to November) growing season obtained after single extraction and 745
sequential extraction, respectively. Error bars correspond to standard deviation (n=4).
746 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Freeze-drying
Without With
Grinding
Milling Mortar & pestle
Liquid nitrogen
Figure1 Click here to download Figure Fig1.pdf
Figure2 Click here to download Figure Figure2.jpg
Figure3 Click here to download Figure Figure3.jpg
Figure4 Click here to download Figure Fig4.png
Table 1: Tested performance parameters of the chromatographic separation.
Performance parameters
Samples Compounds Protocol
(replicates x days)
Acceptance criteria
Results
Selectivity
Spinach
Beech chl a, chl b, β-car Full factorial DoE Selectivity factor >1.3
chl a: 1.4 chl b: 1.5 β-car: 1.2 Application range Standards chl a, β-car 6 concentrations
1r x 2d
Anova lack-of- fit test
P<0.05 (Fig. S) Intra- and inter-
precision
Spinach Beech
chl a, chl b, chl
dev, β-car, car dev 3r x 5d ≤ 10% ≤7.8%
Trueness Standards chl a 2r x 5d ≤ 5% ≤ 1%
LOD Blank chl dev, car dev Signal-to-noise
ratio of 3
LOQ Standards
Spinach chl dev, car dev Signal-to-noise
ratio of 6
Figure captions Click here to download Figure PetibonEtAl_Fig_JCA_vf.pdf
Fig. 1 Sample preparation procedure and its impact on the chlorophyll composition. (a) Selected
preparation steps (light grey boxes) and the relative composition of chlorophylls including chl a (dark grey), chl b (grey), and chl dev (light grey) associated with (a) freeze drying, (b) grinding, (c) with or without liquid nitrogen. Error bars correspond to the standard deviation (n=3).
Fig. 2 Relative abundance of chlorophylls (chl a, chl b, chl dev) and carotenoids (β-car, car
dev, xan dev) in each fraction of the sequential extraction (a), and corresponding chromatogram at 665nm (b, fraction 1 (extracted with acetone: water); c, fraction 2 (extracted with pure acetone); d, fraction 3 (extracted with isopropanol: hexane)).
18 20 22
Fraction 1 Fraction 2 Fraction 3 0
10 20 30 40 50 60
Relative abudance (%)
Sequential extraction b-car car dev chl a chl b chl dev a.
Absorbance (-)
b. Fraction 1
Absorbance (-)
Time d. Fraction 3
Absorbance (-)
c. Fraction 2
Time
Fig. 3 Chromatograms of a spinach sample at 450 nm (a, b) and at 665 nm (c, d)
10 20 30 40 50 10 15 20 25 30
10 20 30 40 50 10 15 20 25 30
chl a
chl b
Absorbance (-)
a chls & xan chls car
chls car & xan unknowns
b
Absorbance (-)
Retention time (min) c
Retention time (min) d
Fig. 4 Relative composition of chlorophylls (chl a, chl b, chl dev) in early, mid-, and late growing season obtained after a single extraction and a sequential extraction. Error bars correspond to standard deviation (n=4).
Table 1: Tested performance parameters of the chromatographic separation.
Performance parameters
Samples Compounds Protocol
(replicates x days)
Acceptance criteria
Results
Selectivity
Spinach
Beech chl a, chl b, β-car Full factorial DoE Selectivity factor >1.3
chl a: 1.4 chl b: 1.5 β-car: 1.2 Application range Standards chl a, β-car 6 concentrations
1r x 2d
Anova lack-of- fit test
P<0.05 (Fig. S) Intra- and inter-
precision
Spinach Beech
chl a, chl b, chl
dev, β-car, car dev 3r x 5d ≤ 10% ≤7.8%
Trueness Standards chl a 2r x 5d ≤ 5% ≤ 1%
LOD Blank chl dev, car dev Signal-to-noise
ratio of 3
LOQ Standards
Spinach chl dev, car dev Signal-to-noise
ratio of 6
Table1 Click here to download Table Table 1.pdf
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Declaration of Interest Statement (Pls upload in word format)
3/28/22, 4:39 PM Proof and Print Reviewer Comments
Characterization of complex photosynthetic pigment profiles in European deciduous tree leaves by sequential extraction and reversed-phase high performance liquid chromatography.
Original Submission
Tatas Hardo Panintingjati Brotosudarmo, Ph.D.
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Recommendation: Major Revisions Overall Manuscript Rating (1 - 100): 78
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The authors reported characterization of plant pigments originating from leaves using RP-HPLC. As already known, the photosynthetic pigments in higher plants are embedded in the pigment-protein complexes PSII and PSI with their LHCs. The type pigments in higher plants are quite conserved. It is quite amusing that the chromatogram indicated a lot of Chlorophyll derivatives. I don't think that the extraction procedures or chromatographic methods were sufficient to overcome the rapid degradation of the pigments. So it is highly doubtful that these pigment derivatives actually exist or occur in a poor sample preparation process. During sample preparation, usually, additives were used to prevent oxidation, but here in the preparation non, please explain! Please check again the purity of standards and reagents were of low purity! Standards and reagents have to be of high purity (equivalent and above 95% purity). I also don't understand why the author has chosen such a long separation, even more than 50 min, while nowadays a shorter time of less than 20 min was employed. Please explain why Fig 3d shows such high background. Please explain the absorbance unit of the chromatograms shown in Fig3! What does (-) mean? Please correct the name of pigments in Appendix A that is not similar to others in the texts. In Appendix C1, only chl a, chl b, and beta-car were found, where were the other cars?
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3/28/22, 4:39 PM Proof and Print Reviewer Comments 2) Disagree
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Manuscript length (including figures, tables, etc.) [1-10] (Please grade from 1 (lowest or not relevant) to 10 (highest).
1) NA 2) 1 3) 2 4) 3 5) 4 6) 5 7) 6 8) 7 9) 8 10) 9 11) 10 Ans: 7
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3/28/22, 4:40 PM Universitas Ciputra Mail - Review reminder for Phytochemistry
Tatas Brotosudarmo <[email protected]>