Halloysite and Laponite Hybrid Pigments Synthesis with Copper Chlorophyll

Round 1

Reviewer 1 Report

This paper aims the improvement of some properties of chlorophyll dye derivative by making hybrid materials with two types of clay. The purpose itself seems interesting and worth for investigation. However, in this paper, the method of data presentation is quite inappropriate so that the reviewer guesses readers are almost impossible to understand what the authors insist.

For example, Figure 1 and Table 2 might possibly indicate the effect of the difference in the preparation conditions on the amount of dye adsorption on the clays, but the values represented in the figure and table do not indicate the amounts of the dye adsorption. Why not the authors directly show the data of the adsorption amount? The manuscript should be constructed in order for readers to understand as easily as possible.

For this reason, in my opinion, this paper cannot be recommended for publication. Instead, the reviewer recommends the authors to reconstruct their manuscript from the first and submit again as a new paper.

Author Response

"Please see the attachment."

The reviewer is right, for these reason we change the way to present our results in order to make easier the aim of our research. We calculate the amount of the dye adsorbed into the nanoclays using the supernatant absorbance and calculate the response as % of dye adsorbed. In addition, we include and improve the explanation of the dye adsorption calculation process. In this way, the readers could easy understand our method efficacy and the differences founded changing the controlled synthesis factors. You can find the explanation at the new version highlighted in blue font. Here I attach the main changes following your advice, thank you for your contribution.

2.2. Synthesis process

The adsorption of chlorophyll dye into the laminar nanoclays was performed using water solvent and the stirring method. The times to incorporate each modifier were selected according to previous studies [32]. In this study, both nanoclays were dispersed at 1800 rpm for 20 h. Laponite dispersions were prepared at 18 g∙L^-1 to avoid gel formation in distilled water. The halloysite nanoclay was heat-treated for 24 h at 220 ºC [33][34] and dispersed in distilled water at 25∙L^-1. In both nanoclays, the dispersion pH was sifted at 3-4 with HCl^- [35][36]. The dye concentration in solutions was 2 ∙ 10⁻³ M. The surface modifiers concentration fell within a range of 0-2% over the nanoclay mass. Dye exchange was performed by stirring at 1800 rpm and room temperature for 4 h, and then at 600 rpm for 20 h. Solvent separation was carried out by centrifuging to obtain the paste-nano-pigment (PNP). Then the PNP was washed by redispersing its paste at 400 rpm for 30 min until the supernatant was clear. All supernatant separated were collected from the beginning to the final washing step and the measurements were made for each sample just before the supernatant collections in order to avoid the dye degradation. While we were washing the samples to ensure that all the non-adsorbed dye was recovered, we keep the separated solutions apart from the light and temperature. We used hermetic containers protected with aluminum. Then the separated supernatant were made up to a known volume to continue with the spectrometer measurements. In addition, thirteen Chlorophyll dilutions from 1∙10^-6[M] to 1∙10^-4[M] were prepared to obtain the Lambert Beer line as spectrophotometer calibration method. Lastly, the PNP was cool-dried by an ALPHA 1-2 LDplus lyophilizer (Martin Christ, Osterode am Harz, Germany) for 24 h.

 

2.3. Characterization

The determination of the amount of dye adsorbed by the nano-clay system allowed us to define the synthesis performance of the process. For this purpose, a UV–Vis transmission spectrophotometer (JASCO V650, Easton, MD, USA) was utilized to measure the dye absorbance (%) in the separate supernatants. Then we calculated the amount of dye adsorbed by nano-clays as a percentage of the initial concentration in the exchange step. That parameter was employed as a response to minimize in the DoE analysis.

 

3.1. Chlorophyll adsorption

All the supernatants separated after the centrifugation step were measured to find the presence of natural colorants to calculate the synthesis performance as the percentage of the dye adsorbed in both nanoclays (Ads(%)). Significant differences were found depending on the synthesis conditions. The variance analysis revealed that the significant factors in the percentage of the dye adsorbed in both nanoclays (Ads(%)) were the mordant and pH interactions (see Table 3). The p-Values in both factors were lower than 0.05. According to Figure 1, when mordant salt was present (MORD=1.0), no differences appeared due to the pH level (from -1.0 to 1.0), and the supernatant was clear and dye adsorption was maximum (≈ 99%) in both structures. However, when no mordant salt was present (MORD= -1.0), pH became a critical factor and had to be acidic (pH=-1.0 [3-4]) to maintain dye adsorption near to 99% in both nanoclay structures. The lowest adsorption percentage (≈ 84%) was found at a natural pH (pH=1.0 [9-7]) with no mordant salt present (MORD= -1.0).

Table 3. Variance analysis for the Percentage of the dye adsorbed under different synthesis conditions by DoE 2^4-1.

 

source	Sum of Squares	f.d.	Medium square	F-Ratio	P-Value
A:pH	201.863	1	201.863	4.72	0.0526
C:MORD	228.084	1	228.084	5.33	0.0414
AC	211.765	1	211.765	4.95	0.0479
Block (nanoclay)	20.7034	1	20.7034	0.48	0.5010
Total Error	470.507	11	42.7734
Total (corr.)	1132.92	15

 

Figure 1. Interactions plot for the synthesis performance as the percentage of the dye adsorbed (Ads (%)) under different with pH and MORD levels.

Author Response File: Author Response.docx

Reviewer 2 Report

This is a very interesting paper and I really like the statistical analysis of data and the selection of experiments using the Design of Experiments approach. 

I have some minor and major comments:

Minor comments:

• Please review English style. In the attached document I have highlighted some English corrections.
• Please review the numbering of tables (including the text), there are two tables with number 2
• Please include the results from the spectophotometer, or your evaluation of dye absorption. Without these values it is not possible to assess if the data in table 2 are correct or not. 

 

Major comments:

Regarding the methodology, I have concerns about the method suggested to evaluate the absorption of dye into the nanoclays. The information provided in this paper is not enough, in my opinion, to assess if this methodology is appropriate for this case. I believe authors should provide a justification for selection this method and more details. During the manufacturing of the nanopigment, authors used 2 drying steps: spinning and freeze-drying. But they only use the supernatant of dispersing the nanopigment after the first drying step (spinning). These are some of my questions to assess the suitability of this method:

• If you want to assess the amount of dye in the final product, why the authors did not use other techniques like element analysis on the final nanopigment?
• How can they be sure that the amount of dye in the supernatant is the one that was not absorbed and that no dye was lost during the spinning (centrifugation) process?
• When did you perform the measurement of the supernatant, after the 22 h of dispersion? Was the dispersion kept away from light? Was the supernatant stored and measured after the manufacturing of all the samples? If so, under what conditions was it kept? Would these conditions could have degraded the dye (as you mentioned it is sensitive to oxygen and light)?

If the authors could answer these questions and clarify these aspects for this method, then I would be happy to accept it.

Author Response

"Please see the attachment." 

Reviwer 2:
This is a very interesting paper and I really like the statistical analysis of data and the selection of experiments using the Design of Experiments approach. 
I have some minor and major comments:
Minor comments:
•    Please review English style. In the attached document I have highlighted some English corrections.
We would like to thank the review for that contribution. We add the corrections and in addition, we have send the document to a Native English person for the revisions (bill attached).
•    Please review the numbering of tables (including the text), there are two tables with number 2
The reviewer is right, we also detected a mistake with table’s 3-4 enumerations and we corrected all of them.
•    Please include the results from the spectophotometer, or your evaluation of dye absorption. Without these values it is not possible to assess if the data in table 2 are correct or not.
We add a new Table 2, with the absorbance measured by the spectrophotometer and the percentage of the adsorbed dye calculated from these data and used as new response to DoE as Reviewer 1 suggested. In additions, as supplementary material, we add the Lambert Beer Line and equation used for the spectrophotometer calibration with the Chlorophyll dye.   
Table 2. Absorption (%) results at  405.4nm, and Percentage of the dye adsorbed under different synthesis conditions by DoE 24-1, calculation. 
REF.    Abs lambda (405,4 nm)    *Ads (%)
GLAP.1    0.1267876    97.91
GLAP.2    0.4166450    80.21
GLAP.3    0.1168471    98.51
GLAP.4    0.1445317    96.82
GLAP.5    0.1220899    98.19
GLAP.6    0.1101210    98.92
GLAP.7    0.1197555    98.34
GLAP.8    0.1242313    98.06
GHA.1    0.1197628    98.34
GHA.2    0.2486290    90.47
GHA.3    0.1279232    97.84
GHA.4    0.6237080    67.57
GHA.5    0.1153758    98.60
GHA.6    0.1140121    98.69
GHA.7    0.1160737    98.56
GHA.8    0.1136496    98.71

Supplementary material: 
Lambert-Beer line equations and R2
Dye    Equation    R2
Chlorophyll    y = 33806x + 0.0925    0.9994
 

Major comments:
Regarding the methodology, I have concerns about the method suggested to evaluate the absorption of dye into the nanoclays. The information provided in this paper is not enough, in my opinion, to assess if this methodology is appropriate for this case. I believe authors should provide a justification for selection this method and more details. During the manufacturing of the nanopigment, authors used 2 drying steps: spinning and freeze-drying. But they only use the supernatant of dispersing the nanopigment after the first drying step (spinning). These are some of my questions to assess the suitability of this method:
•    If you want to assess the amount of dye in the final product, why the authors did not use other techniques like element analysis on the final nanopigment?
•    How can they be sure that the amount of dye in the supernatant is the one that was not absorbed and that no dye was lost during the spinning (centrifugation) process?
•    When did you perform the measurement of the supernatant, after the 22 h of dispersion? Was the dispersion kept away from light? Was the supernatant stored and measured after the manufacturing of all the samples? If so, under what conditions was it kept? Would these conditions could have degraded the dye (as you mentioned it is sensitive to oxygen and light)?
If the authors could answer these questions and clarify these aspects for this method, then I would be happy to accept it.
The reviewer is right we did not provide enough information to clarify the method used to calculate the dye adsorbed into the nanoclay. We improve the explanation in the synthesis process section, answering all the questions as you can see below. We really thank your contribution to improve the quality of our work. 
Then the PNP was washed by redispersing its paste at 400 rpm for 30 min until the supernatant was clear. All supernatant separated were collected from the beginning to the final washing step and the measurements were made for each sample just before the supernatant collections in order to avoid the dye degradation. While we were washing the samples to ensure that all the non-adsorbed dye was recovered, we keep the separated solutions apart from the light and temperature. We used hermetic containers protected with aluminum. Then the separated supernatant were made up to a known volume to continue with the spectrometer measurements. In addition, thirteen Chlorophyll dilutions from 1∙10-6[M] to 1∙10-4[M] were prepared to obtain the Lambert Beer line as spectrophotometer calibration method. Lastly, the PNP was cool-dried by an ALPHA 1-2 LDplus lyophilizer (Martin Christ, Osterode am Harz, Germany) for 24 h.

Author Response File: Author Response.docx

Reviewer 3 Report

The subject of the presented paper was focused on the development of the hybrid pigment synthesis method.

Although the description of the results is very extensive, however, I have some doubts about the research conception. As I guess, the main goal of the research is to develop process-stable pigments. In the discussed case, the desired color was green, which the authors intended to obtain by modifying the chlorophyll.

Until this point the research description is clear to me, however, there is no clear information on the practical results of the work hereafter.

The description of the results is only a report on the course of measurements. The subject of the article is pigment production and there is no photo of the sample in the work to prove obtained effects.

The conclusions are therefore not supported by any analysis of the stability of the obtained samples.

In the current shape, I recommend supplementing the research with results confirming the coloring ability in relation to the base nanoclays.

Author Response

Reviewer 3:

The subject of the presented paper was focused on the development of the hybrid pigment synthesis method.

Although the description of the results is very extensive, however, I have some doubts about the research conception. As I guess, the main goal of the research is to develop process-stable pigments. In the discussed case, the desired color was green, which the authors intended to obtain by modifying the chlorophyll.

Until this point the research description is clear to me, however, there is no clear information on the practical results of the work hereafter.

The description of the results is only a report on the course of measurements. The subject of the article is pigment production and there is no photo of the sample in the work to prove obtained effects.

The reviewer was right the goal of the research was to produce interesting stable natural hybrid pigments. For this reason, we provide more evidences of the results including pictures about the green powder that we obtained as you can see in new Figure X. In addition, we decided to make an application using an epoxy bioresin, in order to follow with the green chemistry philosophy of our work as we describe in the new section “Application” and you can find in new Figure X.

The conclusions are therefore not supported by any analysis of the stability of the obtained samples.

The reviewer was right. We add a stability test in order to ensure the color fastness for the hybrid natural pigments. As you can see in the new results from the Xenon Test analysis both nanoclays provide a stabilization against the solar exposures and the color differences that we observed were due to the bioresin degradation while the green powder remains dark and green as we commented in our new conclusions.

In the current shape, I recommend supplementing the research with results confirming the coloring ability in relation to the base nanoclays.

We would like to thank the reviewer for the suggestion. We add the new results following his/her comments and increase the interest of the presented work as you can see in the added parts:

Abstract: Sustainable… Finally, a wide chromatic green color range was obtained with the surfactant modification in both nanoclays, and the colour fastens was also improved in the hybrid pigments application. The samples generated with 10% of hybrid pigments from both nanoclays and an Epoxy bioresin, show higher colourfastness than the sample with the natural chlorophyll, due to the nanoclays-dye interaction and protection.  

2.1. Materials

Lastly, for bio-nanocomposite generation, we used the bioresin whose trade name is GreenPoxy 55, which is an epoxy system with a single hardener where 55% of the molecular structure is of plant origin. Catalyst SD 505 came from SICOMIN Composites (Chateauneuf les Martigues, France).

2.2. Synthesis process

Lastly, the PNP was cool-dried by an ALPHA 1-2 LDplus lyophilizer (Martin Christ, Osterode am Harz, Germany) for 24 h providing the fine green powder that we used for the characterization and the application (Figure 1).

Figure 1. Synthesis steps pictures with halloysite (GHA) and laponite (GLAP) examples.

2.3. Biocomposite Generation

Bio-composite materials were handmade by mixing with the commercially recommended catalyst, and using silicon templates to obtain plain rectangular samples (Figure 2). We employed 10% over the bio-resin mass nano-pigment concentrations. The curing process was carried out at 90 °C for 3 h.

Figure 2. Biocomposite preparation after epoxy 55 curing step with GHA and GLAP hybrid pigment addition at diferent synthesis conditions from [1-8].

2.4. Characterization

Finally, a SOLARBOX 1500e RH climatic chamber (ERICHSEN, Hemer, Germany) was used to measure color UV–Vis fastness. All samples were measured using a Konica Minolta sphere integrated spectrophotometer (CM-2600d) to obtain the reflectance factors r(l)[370-740] nm range with the D65 illuminant and the CIE-1964 standard observer. The measurements were made at several exposure time intervals, and color differences were calculated by measuring samples before and after radiation exposure. Color differences DE_ab* were calculated with the colorimetric attributes of the CIELAB color space.

Table 4. Analysis of variance for C_ab* values for the different synthesis conditions by DoE 2^4-1.

Table 5. Analysis of variance for L* values for the different synthesis conditions by DoE 2^4-1.

3.5. Colour fastness

The aging test was controlled by taking color measurements from t0 (initial) using the samples with the 10% hybrid pigment or the original dyes, to 102 hours, which corresponds to three months with real sun exposure. Previous works have checked organic dye stabilisation in nanoclay structures by changes in absorption curves [45]. We calculated the colour difference (DEab*), and standardised the calculated values by natural dye content (g) in each sample (DEab*/g.dye). Standard errors were also calculated and represented to make comparisons. Figure 16 shows, the color UV–Vis fastness of chlorophyll natural dye increased when used as hybrid pigment with both nano-clays. The color differences calculated as DEab* (g.col) were significantly bigger for the sample with the original natural dye. For 22 hours, the color differences of the samples with the original dye was bigger than 4 DEab* units, which corresponds to a marked visible change for the human eye, meanwhile the hybrid samples keep the colour differences under 1 DEab* units (Figure 16). It is also remarkable that the colour differences that we could observe in the biocomposites whit both nanoclays were a yellowish due to the epoxy resin degradation. However, in the sample generated with the natural chlorophyll the color difference corresponds to a significant dye degradation with lighter appearance.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I have already made a decision that this paper should be rejected. I have commented that the authors should submit again as a NEW paper. Therefore, I am quite surprised to see that the revised version is submitted and handed to me for a review. In the previous comment, I did not mean that only Figure 1 and Table 2 should be revised, but that almost ALL data including figures and tables should be revised because they are hard for readers to understand. Such revisions must be far beyond the ordinary revision, which means constructing a new paper. The authors should have reconstructed their manuscript from the first.

As for Figure 1 and Table 2, the revisions made on this version could partly be appropriate (I still doubt the necessity of introducing the parameters of MORD(1) and pH(-1) etc.). But this paper can no more be reviewed, as I believe that authors now fully understand my intension.

Author Response

The reviewer is right; we not fully understand their comments. We are sorry and would like to invite the reviewer to reconsider his/her opinion taking into account the following highlights:

• We understand that at first it was hard to understand the DoE analysis using and indirect parameter as response, because the interpretation was not direct. However, making the calculations about the dye adsorbed (%) into the nanoclays, the interpretation is easier and the conclusions are direct.
• Ion strength and pH are important factors to control the adsorption process that involves the nanoclay and organic compounds as it was previously found[1][2][3]. For this reason is important to ad those parameters as control factors to ensure that the synthesis conditions are optimal to adsorb the maximum amount of the dye. In addition this (Mordant salt and pH) and the other synthesis factor that we selected, could affect the optical and physical properties as we have shown.
• Other reason to combine different synthesis factors: based on the characteristics of structure and surface charge of the halloysite nanoclay, the common modification techniques proposed mainly include thermal treatment, acid washing, pillaring, organic modification, etc. These strategies have their own unique advantages. Therefore, the adsorption limitations of clays are gradually broken down with incremental improvements by research. Some studies also combine two or more of these methods, as we did [4], in order to obtain multifunctional adsorbents, such as simultaneous uptake of organic and inorganic contaminants using inorgano-organo-clay.
• Other recent studies combine different hybrid properties in their studies to characterize hybrid compounds as the ones that we presented. Aydin Hassani et al. highlighted that they make a comparative adsorption study, using statistical design of experiments, and then they characterize the hybrids using different techniques as SEM, EDX, XRD, BET and FTIR [5]. Emmanuel Nyankson et.all, presents the effects of Ag₂CO₃-halloysite composites on the efficient removal of water soluble dyes, combining XRD, FTIR, SEM, TGA curves, and Reflectance values (%), without a color perception calculations [6] as we did to improve the optical characterization of the final hybrid pigments.
• Despite the reviewer said that “ALL data including figures and tables should be revised because they are hard for readers to understand”, in the previous cited examples we had found the same data presentation and discussion. Is true that there are a lot of works which does not include the statistical design (for example; [7]), but this is a weakness not an strength. We hardly believe that the means plots and interactions plots could be hard to follow at the beginning, but then provides reliable and complete information, as the ANOVA tables that complements this information.
• The CIELAB parameters has also been previously used, and some authors just used table to discuss the measured data [8]. We improve the presentation of our result including the diagrams precisely to make easier for the readers to compare the optical properties of all the hybrid pigments.
• Finally, our XRD patterns, which includes zoom in the discussed parts, and the TGA analysis have high resolution, and are not difficult to read, so we could not understand any problem with their interpretation, as previous works did with this nanoclay [9] [10].

Taking into account our discussion, and the changes that we has made before (attached again) we hope that the reviewer finally found our work suitable for publication.

 

References

1. Ngulube, T.; Gumbo, J. R.; Masindi, V.; Maity, A. Evaluation of the Efficacy of Halloysite Nanotubes in the Removal of Acidic and Basic Dyes from Aqueous Solution. Clay Miner. 2019, 54 (2), 197–207.
2. Salam, M. A.; Kosa, S. A.; Al-Beladi, A. A. Application of Nanoclay for the Adsorptive Removal of Orange G Dye from Aqueous Solution. J. Mol. Liq. 2017, 241, 469–477.
3. Dey, D.; Bhattacharjee, D.; Chakraborty, S.; Hussain, S. A. Effect of Nanoclay Laponite and PH on the Energy Transfer between Fluorescent Dyes. J. Photochem. Photobiol. A Chem. 2013, 252, 174–182.
4. Lee, S. M.; Tiwari, D. Organo and Inorgano-Organo-Modified Clays in the Remediation of Aqueous Solutions: An Overview. Appl. Clay Sci. 2012, 59, 84–102.
5. Hassani, A.; Khataee, A.; Karaca, S.; Karaca, M.; Kıranşan, M. Adsorption of Two Cationic Textile Dyes from Water with Modified Nanoclay: A Comparative Study by Using Central Composite Design. J. Environ. Chem. Eng. 2015, 3 (4), 2738–2749.
6. Nyankson, E.; Agyei-Tuffour, B.; Annan, E.; Yaya, A.; Mensah, B.; Onwona-Agyeman, B.; Amedalor, R.; Kwaku-Frimpong, B.; Efavi, J. K. Ag2CO3-Halloysite Nanotubes Composite with Enhanced Removal Efficiency for Water Soluble Dyes. Heliyon 2019, 5 (6), e01969. https://doi.org/10.1016/J.HELIYON.2019.E01969.
7. Golubeva, O. Y.; Alikina, Y. A.; Kalashnikova, T. A. Influence of Hydrothermal Synthesis Conditions on the Morphology and Sorption Properties of Porous Aluminosilicates with Kaolinite and Halloysite Structures. Appl. Clay Sci. 2020, 199, 105879.
8. Sadegh-Hassani, F.; Nafchi, A. M. Preparation and Characterization of Bionanocomposite Films Based on Potato Starch/Halloysite Nanoclay. Int. J. Biol. Macromol. 2014, 67, 458–462.
9. Szczepanik, B.; Słomkiewicz, P.; Garnuszek, M.; Czech, K.; Banaś, D.; Kubala-Kukuś, A.; Stabrawa, I. The Effect of Chemical Modification on the Physico-Chemical Characteristics of Halloysite: FTIR, XRF, and XRD Studies. J. Mol. Struct. 2015, 1084, 16–22.
10. Asgar, H.; Jin, J.; Miller, J.; Kuzmenko, I.; Gadikota, G. Contrasting Thermally-Induced Structural and Microstructural Evolution of Alumino-Silicates with Tubular and Planar Arrangements: Case Study of Halloysite and Kaolinite. Colloids Surfaces A Physicochem. Eng. Asp. 2021, 613, 126106. https://doi.org/https://doi.org/10.1016/j.colsurfa.2020.126106.

 

Author Response File: Author Response.docx

Reviewer 2 Report

I'm happy with the changes made to this manuscript. 

I recommend English revision of the new content (page 3, lines 109 - 117). This is my suggestion for correction of the section:

All separated supernatant was collected from the beginning to the final washing step. The measurements were made for each sample just before the supernatant collections in order to avoid the dye degradation. To ensure that all the non-adsorbed dye was recovered, water and solution from washing were kept protected from light and temperature in hermetic containers covered with aluminium.

Author Response

We want to thank the reviewer for the suggestions. In addition, we send again the text for their correction as you can see in the following images. Thank you very much for your comments. The final revised text is:

All separated supernatant was collected from the beginning to the final washing step. The measurements were made for each sample just before the supernatant collections in order to avoid the dye degradation. To ensure that all the non-adsorbed dye was recovered, water and solution from washing were kept protected from light and temperature in hermetic containers covered with aluminium. Then the separated supernatant were made up to a known volume to continue with the spectrometer measurements. In addition, thirteen Chlorophyll dilutions from 1∙10^-6[M] to 1∙10^-4[M] were prepared to obtain the Lambert Beer line as spectrophotometer calibration method. Lastly, the PNP was cool-dried by an ALPHA 1-2 LDplus lyophilizer (Martin Christ, Osterode am Harz, Germany) for 24 h.

 

2.3. Characterization

The determination of the amount of dye adsorbed by the nano-clay system allowed us to define the synthesis performance of the process. For this purpose, a UV–Vis transmission spectrophotometer (JASCO V650, Easton, MD, USA) was utilized to measure the dye absorbance (%) in the separate supernatants. Then we calculated the amount of dye adsorbed by nano-clays as a percentage of the initial concentration in the exchange step. That parameter was employed as a response to minimize in the DoE analysis.

See attached document:

Figure 1. Revision answer 21/05/2021

Figure 2 Invoice for English revision

 

Author Response File: Author Response.docx

Reviewer 3 Report

Most of the reviewers comments has been taken into account in the newest version of the manucript. I suggest publication in the current form.