Aqueous Two-Phase Systems Based on Ionic Liquids and Deep Eutectic Solvents as a Tool for the Recovery of Non-Protein Bioactive Compounds—A Review

Round 1

Reviewer 1 Report

1)All parts of the manuscript (abstract, Bioactive Compounds, Methods for Obtaining Bioactive Compounds and Bioseparation, ……. Conclusion) should be more quantitative,

2)Please highlight the novelty of this study in your manuscript,

3)Exprimental conditions should be added to table 1…Table 5,

4)Add nomenclature to the manuscript,

5)What are your important recommandations by the end of this study,

6)This maniscript needs to be formatted carefully and the language should be re-approved by a native speaker/Grammarly checked.

Author Response

1) All parts of the manuscript (Abstract, Bioactive Compounds, Methods for Obtaining Bioactive Compounds and Bioseparation, ……. Conclusion) should be more quantitative

We understand the reviewer's suggestion. The current manuscript is a review article about the application of non-conventional Aqueous Two-Phase Systems (ATPS) for extraction, recovery, purification, and concentration of non-protein bioactive compounds. This review addresses more qualitative than quantitative questions regarding the topic under study due to the difficulty of comparing results from the separation process. Some reasons related to this include different methods for obtaining the most varied types of bioactive compounds, the complexity of the raw matrices, and the different compositions of aqueous systems combining ionic liquids, eutectic and organic solvents. In addition, data regarding the purification process are scarce since some studies only use the technique to purify the target bioactive compound and are not focused on studying the aqueous-phase system itself. Thus, the expression and comparison of quantitative data in terms of extraction yields, concentration factor, and purity are compromised. On the other hand, this review presents a wide range of possibilities for applying non-conventional ATPS to bioactive compounds, its main aspects and critical factors, and future perspectives on this theme.

 

2) Please highlight the novelty of this study in your manuscript

We appreciate the reviewer's suggestion. We highlighted the novelty of the study at the end of Introduction section (page 3), as follows:

“In this sense, this review aimed to demonstrate the application of ATPS based on IL and DES as a tool for the recovery of non-protein bioactive compounds, considering important factors for the application, main results, and most recent advances in this field. To the best of our knowledge, this is the first time that a literature review explores non-conventional ATPS applied to a wide range of compounds of non-protein origin.”

 

3) Experimental conditions should be added to Table 1…Table 5

We appreciate the reviewer's suggestion. All tables present essential information about different types of non-conventional ATPS that can be applied to extract and recover non-protein bioactive compounds. Since the compounds differ in type, raw material complexity, bioactivity, and mode of production, the experimental conditions are also different and not comparable. For this reason, the overall experimental conditions were not included in the tables. Likewise, the reasons for applying ATPS – extraction or purification of bioactive compounds – also differ between compounds, which limits the presentation of experimental data other than the general composition of phase-forming aqueous systems. Thus, we understand that the purpose of these tables is to present, in a general way, the possibilities of applying non-conventional ATPS in non-protein bioactive compounds. The availability of information such as these and in a compiled form is still scarce and more directed to traditional ATPS and compounds of protein origin.

 

4) Add nomenclature to the manuscript

We appreciate the reviewer's suggestion. We added a list of nomenclature to the end of the manuscript.

 

 

 

5) What are your important recommandations by the end of this study

  Traditional ATPS has been widely explored in the scientific literature, mainly in protein recovery and purification. However, studies focused on non-conventional ATPS can be limited, mainly when applied to separating non-protein bioactive compounds extracted or obtained from complex matrices or systems. In this sense, we recommend future investigations focused on constructing more efficient non-conventional ATPS to separate different types of compounds. We recommend more studies focused on phase-forming non-conventional ATPS processes and establishing optimal operational parameters. In line with this, molecular simulation as a pre-screening tool can also be promising for expanding non-conventional ATPS in separating and purifying bioactive compounds.

Additionally, we understand that it is necessary to expand the quantity and diversity of compounds used in the construction of non-conventional ATPS, providing greater versatility and options for suitable application in recovery and purification processes. Regarding bioactive compounds, the increase, maintenance, or loss of their bioactivities must be verified after separation by non-conventional ATPS, thus ensuring the viability and functionality of these compounds in their future applications. All these points were highlighted in the topic "7 – Conclusion and future perspectives" (page 28).

 

 6) This manuscript needs to be formatted carefully and the language should be re-approved by a native speaker/grammarly checked.

We appreciate the reviewer's recommendation. The English language of the manuscript was revised entirely by native speakers (see the certification attached to the submission).

Author Response File: Author Response.pdf

Reviewer 2 Report

1- The Abstract is somewhat dry; it should include more actual results from the manuscript.
2- The Authors should also consider rephrasing some of the paragraphs, to improve the manuscript in terms of the English language.
3- Could the authors compare their results with the properties of the other materials?

Author Response

1) The Abstract is somewhat dry; it should include more actual results from the manuscript.

We appreciate the reviewer's suggestion. We modified the abstract in order to bring the main results/conclusions of this review article.

2) The Authors should also consider rephrasing some of the paragraphs, to improve the manuscript in terms of the English language.

We appreciate the reviewer's recommendation. The English of the manuscript was revised entirely.

 

3) Could the authors compare their results with the properties of the other materials?

             We appreciate the reviewer's suggestion. The current manuscript is a review article about the application of non-conventional ATPS for extraction, recovery, purification, and concentration of non-protein bioactive compounds. Due to the wide range of possibilities for obtaining these compounds, raw material complexities, different classes of bioactive compounds, and distinct composition of aqueous systems combining ionic liquids and eutectic and organic solvents, the comparison of results become problematic. In addition, some studies only use ABTS to obtain the bioactive compound, while others evaluate the composition of aqueous systems and operational parameters. Due to these differences, this review does not address the comparison in terms of extraction yields and concentration and purification factors. However, it presents an overview of the possibilities of applying non-conventional ATPS to bioactive compounds.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments on processes-2041538:

 

The current review as a critical addition to literature would benefit the field of ionic liquids derivatives. Before acceptance, modifications considering the following points would be sufficient.

 

Biphasic separation, also known as partition or extraction, is a critical application of ionic liquids derivatives. Aside from the existing literature overview, a worth noting point to add is the usefulness of molecular simulation. The simulation tool, which historically serves as a complementing tool to experimental measurements, already grows to be a practically usable instrument of predictive power. Recent efforts on extensive free energy calculations for molecular solvation and partition enable the summarization of near-optimal modelling protocols that ensure accurate calculations of bulk properties and solvation/partition thermodynamics simultaneously. For example, in J. Mol. Liq., 2022, 120492, J. Ion. Liq., 2022, 2, 100043, and Adv. Theory Simul., 2022, 5, 2200274, the solid numerical evidence proves the applicability of the recommended modelling procedure to most ionic liquids. Some of the modelled ionic liquids in the mentioned papers are covered by the 2D chemical structures presented in Figure 2 of the current manuscript, e.g., imidazolium and phosphonium cations and dicyanamide anion. With the recommended modelling procedure, even for newly encountered ionic liquids derivatives, the solvation and partition thermodynamics can be accurately estimated with computational tools, without experimental data.

 

Many abbreviations are defined but are not used, e.g., AIL, RV and TL. In that case, they should not be defined at all.

Author Response

1) Biphasic separation, also known as partition or extraction, is a critical application of ionic liquids derivatives. Aside from the existing literature overview, a worth noting point to add is the usefulness of molecular simulation. The simulation tool, which historically serves as a complementing tool to experimental measurements, already grows to be a practically usable instrument of predictive power. Recent efforts on extensive free energy calculations for molecular solvation and partition enable the summarization of near-optimal modelling protocols that ensure accurate calculations of bulk properties and solvation/partition thermodynamics simultaneously. For example, in J. Mol. Liq., 2022, 120492, J. Ion. Liq., 2022, 2, 100043, and Adv. Theory Simul., 2022, 5, 2200274, the solid numerical evidence proves the applicability of the recommended modelling procedure to most ionic liquids. Some of the modelled ionic liquids in the mentioned papers are covered by the 2D chemical structures presented in Figure 2 of the current manuscript, e.g., imidazolium and phosphonium cations and dicyanamide anion. With the recommended modelling procedure, even for newly encountered ionic liquids derivatives, the solvation and partition thermodynamics can be accurately estimated with computational tools, without experimental data.

We appreciate the reviewer's suggestion. A topic focused on molecular simulation was added to the manuscript (page 16, topic 5 – Computational modeling related to ATPS and applied to bioactive compounds). We also added some information about molecular modeling was added in the “Introduction” and “Conclusion and Future Perspectives” sections, as follows:

Introduction (page 2): “Advances in this field, including molecular modeling of IL and DES, could facilitate the expansion and development of purification systems through model prediction, especially when it comes to new and poorly studied alternative solvents, thus also contributing to overcoming time and money consumption.” 

Conclusion and Future perspectives: “In line with this, molecular simulation as a pre-screening tool can also be promising for expanding non-conventional ATPS in separating and purifying bioactive compounds.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Manuscript improved. 

Reviewer 3 Report

 Acceptable in the current form.