Turning a 3D Printer into a HPLC Fraction Collector: A Tool for Compound-Specific Stable Isotope Measurements

Round 1

Reviewer 1 Report

The authors repurposed a commercial 3D printer for use as fraction collector and simultaneous solvent evaporator. Methodology is very well detailed and allows for replication of the design and experiments. Experimental setup is clever and the paper is well presented and nice to read.
Two considerations:
- In the proof of concept, the authors use MilliQ water. But all the design in tin capsules was also thought to accomodate for organic and/or corrosive samples. Since this constitutes a risk, is the design appropriate and safe to handle solvent evaporation? is the collector able to be placed under a hood, or isolated to convey vapours elsewhere? this deserves space in the discussion.
- likewise, microvolume centrifuge tubes ("Eppendorf") are also suited for organic solvents and can fit in the racks, allowing for easier handling of collected material.

Author Response

Thank you for the positive comments. Responses to enquiries are below:
- In the proof of concept, the authors use MilliQ water. But all the design in tin capsules was also thought to accommodate for organic and/or corrosive samples. Since this constitutes a risk, is the design appropriate and safe to handle solvent evaporation? is the collector able to be placed under a hood, or isolated to convey vapours elsewhere? this deserves space in the discussion.
R – We thank the reviewer for this suggestion. We have amended the manuscript to address this issue (lines 381 to 386).
-likewise, microvolume centrifuge tubes ("Eppendorf") are also suited for organic solvents and can fit in the racks, allowing for easier handling of collected material.
R - We also thank the reviewer for this suggestion. We have amended the manuscript to include this potential adaptation of our approach (lines 387 to 390).

Reviewer 2 Report

Dear Authors,

Your work on the development of a low-cost fraction collector interfaced with an HPLC for compound-specific stable isotope analysis is intriguing. However, certain aspects could benefit from further explanation to enhance the manuscript's clarity and depth.

Please consider providing additional details about the HPLC setup, the rationale behind the glucose solution concentration, and a more comprehensive explanation of the fraction collector's advantages over existing systems. Furthermore, elaborating on the system's compatibility with different 3D printer models and insights into potential enhancements would significantly enrich the manuscript.

Additionally, elaborating on the impact of different flow rates on glucose collection and further insight into potential challenges and their mitigation strategies would greatly strengthen the paper's contribution.

Your attention to these aspects will significantly enhance the paper's value and potential for publication.

1.       Could you elaborate more on the specifics of the HPLC setup, particularly on the column choice, temperature settings, and any particular considerations made to prevent column clogging?

2.       What was the rationale behind choosing 4 mM as the final concentration for the glucose solution? Was there any particular significance to this concentration in terms of the experimental objectives?

3.       The impact of different flow rates on glucose collection is discussed. Can you detail further how the collection time was optimized at different flow rates, and why was 0.6 mL min-1 preferred over 0.3 mL min-1?

4.       How was the accuracy of the fraction collector measured? Was the consistency in measurements across multiple trials assessed thoroughly?

5.       Could you elaborate on the specific advantages of this low-cost fraction collector compared to existing systems? In what ways does it improve or offer distinctive features in compound-specific sample preparation for stable isotope analyses?

6.       The adaptability of the fraction collector to different 3D printer models is mentioned. Are there any specific requirements or constraints for a 3D printer to function effectively as a fraction collector? Additionally, any insights on the durability of this system for extended operation would be beneficial.

7.       The need for a detector in the HPLC for optimized collection times is mentioned. Could you further explain how the addition of a detector could improve the system's performance?

8.       In addressing the challenges associated with peak spreading and co-elution of substances, could you discuss potential strategies or developments that might mitigate these issues in the future?

Author Response

Thank you for the detailed review of our manuscript. Responses to the enquires below.

1 - Could you elaborate more on the specifics of the HPLC setup, particularly on the column choice, temperature settings, and any particular considerations made to prevent column clogging?

R – This is explained in section 5.1, first paragraph (lines 326 to 332). 

2) What was the rationale behind choosing 4 mM as the final concentration for the glucose solution? Was there any particular significance to this concentration in terms of the experimental objectives?

R - This solution was chosen simply because it was concentrated enough to allow a quick demonstration of the use of the setup. This is now described in section 5.1 (lines 334 to 336).

3) The impact of different flow rates on glucose collection is discussed. Can you detail further how the collection time was optimized at different flow rates, and why was 0.6 mL min-1 preferred over 0.3 mL min-1?

R – Explanations about the optimization of the collection time are given in lines 340 – 346). 

4) How was the accuracy of the fraction collector measured? Was the consistency in measurements across multiple trials assessed thoroughly?

R – Please see lines 346-357.

5) Could you elaborate on the specific advantages of this low-cost fraction collector compared to existing systems? In what ways does it improve or offer distinctive features in compound-specific sample preparation for stable isotope analyses?

R - Some discussion about this in lines 368-371, some more points related to this topic in the "Conclusions" section as a whole.

6) The adaptability of the fraction collector to different 3D printer models is mentioned. Are there any specific requirements or constraints for a 3D printer to function effectively as a fraction collector? Additionally, any insights on the durability of this system for extended operation would be beneficial.

R - Lines 368-371 comment about the suitability of 3D printers as fraction collectors. Lines 372-375 deal with the potential durability of this system.

7) The need for a detector in the HPLC for optimized collection times is mentioned. Could you further explain how the addition of a detector could improve the system's performance?

R – Further details on the ability of a detector to improve the system have been incorporated into the manuscript (lines 391-398).

8) In addressing the challenges associated with peak spreading and co-elution of substances, could you discuss potential strategies or developments that might mitigate these issues in the future?

R - We think this is beyond the scope of this paper. These are intrinsic problems of the techniques of liquid and gas chromatography, and have been extensively discussed in the literature for decades. New columns, different solvents, different temperature regimes, different separation columns, redundant columns, HPLC followed by HPLC, or by gas chromatography, etc., are all solutions for these problems, and each one by itself is sometimes deserving of a full research paper. The reference we cite in line 401 describes some of these challenges and how to approach them.

Reviewer 3 Report

The manuscript

"Turning a 3D printer into a HPLC fraction collector: a tool for compound-specific stable isotope measurements"

represents a comprehensive and solidly presented study that is innovative and relevant for the field of Analytical Chemistry. I recommend publication.

Other comments:

1. What is the main question addressed by the research?    

>> Using a 3D printer to build a HPLC fraction collector for compound-specific stable isotope measurements

2. Do you consider the topic original or relevant in the field? Does it
address a specific gap in the field?

>> The aim of using 3D printers to build instruments for Analytical Chemistry is original and innovative. It opens many opportunities for low-cost chemical analysis and to democratize them.

3. What does it add to the subject area compared with other published
material?

>> This manuscript is innovative on its own but adds value compared to other compound-specific stable isotope measurement techniques.

4. What specific improvements should the authors consider regarding the
methodology? What further controls should be considered?

>> As a proof-of-concept study, I think, the authors did a good job. Obviously, one might think of many improvements, as for any analytical instrument, but I have no further concrete recommendations at this stage of development.

5. Are the conclusions consistent with the evidence and arguments presented
and do they address the main question posed?

>> Yes, supported by the validation part.

6. Are the references appropriate?    

>> Yes.

7. Please include any additional comments on the tables and figures.   

>> The figures are less of "scientific nature" but more of a technical description but I think, this is suitable for the Journal Hardware.

Author Response

Thanks for the many positive comments.

Reviewer 4 Report

In this study, the authors presented a HPLC fraction collector as a simple adaptation of a 3D printer which can be a tool for compound-specific stable isotope measurements. The proposed technical solution is simple and seems to be able to be used in practice. The structure of the article is well organized and well balanced. The discussion was well described, research results properly interpreted and presented. I have only some remarks and recommendations as follows:

The authors may add in the discussion more informations about any limitations and further developments of the proposed technical solution. 

Missing supplementary material listed in manuscript: Video S1: FractionCollector.mp4. Is the same as on the authors' provided website https://youtu.be/uQRLlZQone4. ?

There are some typos in the text. The text of manuscript should be carefully checked.

Author Response

Thank you for the positive view of the manuscript. Responses below.
 
1) The authors may add in the discussion more information about any limitations and further developments of the proposed technical solution. 
R - This was done as a response to the other reviewers, who suggested several additions to the text. Hopefully this is satisfactory for the reviewer. Please see text highlighted in red in the manuscript, it contains the response to the other reviewers more specific comments.
2) Missing supplementary material listed in manuscript: Video S1: FractionCollector.mp4. Is the same as on the authors' provided website https://youtu.be/uQRLlZQone4. ?
R - Yes, we could not upload the file as supplementary material for some reason, but it would be the same as the youtube video. I believe the editorial team of the journal will fix that at the appropriate stage.
3) There are some typos in the text. The text of manuscript should be carefully checked.
R - We have re-read the manuscript with great care and corrected all errors found.

Round 2

Reviewer 2 Report

Dear Authors,

I trust this message finds you well. I want to express my sincere gratitude for your prompt and thorough response to the reviewer comments on your manuscript. Your dedication to addressing the concerns and making necessary improvements has significantly enhanced the quality of your work.

I am pleased to acknowledge that your revisions have effectively addressed the questions and suggestions raised during the review process. Your commitment to improving the manuscript is commendable and greatly appreciated. The clarity and precision with which you have incorporated the changes contribute to the overall strength of your research.

I believe that your efforts have strengthened the manuscript, and I look forward to seeing it progress through the publication process. Thank you for your diligence and collaborative spirit throughout this review process.