Contributions to the Characterization of Chromogenic Dyes in Color Slides

Round 1

Reviewer 1 Report

The paper “Contributions to the Characterization of Chromogenic Dyes in Colour Slides” presents a very interesting study entailing a multi analytical approach based on different spectroscopic and chromatographic techniques to outline suitable procedures for the characterization of chromogenic dyes. The work is very well contextualized with the literature and quite each step and results thoroughly described and discussed.

However, I think that the paper is too long and not concise with the result of not focusing the attention on the aims of the work. I would suggest shortening the introduction, merging some figures/moving to Supplementary Materials to make the reading more fluent. Consider using Supplementary Materials.

Even if the results are only preliminary, lacking the analysis of reference materials for a proper identification of the dye, the work fill a gap of knowledge on the topic and deserve to be published after minor revisions.

 

Some comments are reported below:

 

HPLC-DAD-MS Abstract:

Replace RH and T with relative humidity (RH) and temperature.

 

Introduction:

The introduction is very detailed, history, typology and technical features of chromogenic dyes are reported denoting a very attentive study of the literature. Nevertheless, I would suggest being more concise, shortening a bit the text to better focalize the attention on the more important information for the comprehension of the paper.

Replace “its” with “theirs” in “Different functional groups can be attached to the colour coupler nucleus to improve its features”.

 

Material and techniques

2.1. Case studies:

Remove are in the sentence “Both are 35 mm chromogenic reversal films are from the integrated dye couplers type”.

I suggest moving Figure 2 in the Supplementary Materials.

2.6. Thin-Layer Chromatography (TLC)

Reformulate the sentence as follows: “Both mixtures from the EPT and RXP were applied on the TLC plate, by putting several drops (ca. 5) on the same spot at the bottom of the TLC plate…”

2.8 and 2.9

I would suggest replacing “linked to” with “coupled with” when referring to “High Performance Liquid Chromatography linked to Diode Array Detection” in all the text.

Why did you use three different chromatographic systems? For comparing the potentialities of each instrumentation? Would the use of HPLC-DAD be avoided in favor of that of HPLC-DAD-MS only? This point needs to be better clarify.

Are the chromatographic conditions adopted for HPLC-DAD-MS the same of LC-HRMS? If yes, I would suggest describing the two instrumentations and then inserting the paragraph relative to the chromatographic conditions (specifying that it is relative to both of them).

Why did you not use the same chromatographic conditions for HPLC-DAD and HPLC-DAD-MS and LC-HRMS?

Is the elution program (and mobile phases) used for HPLC-DAD already optimized for the analysis of dyes? Also, on the basis of the results reported in Section 3.5 (and also as declared by the authors themselves), the conditions used are not optimal (compounds eluted at high retention time and not well separated). Why were any other conditions tested?

Please replace “flow rate of 300 μL min-1” with “flow rate of 0.3 mL min-1” to be consistent with the same unit of measure used for the flow rate of HPLC-DAD.

 

3. Results and discussion

3.1. Optical Microscopy

Reformulate the sentence “By observing the images obtained with the OM, it can be concluded that the film has a thickness of about 130 μm” as “The film thickness is about 130 μm”.

I would suggest merging Figure 3 and Figure 4 in only one Figure (a-d). The same for Figure 5 and Figure 6.

Replace “colon” with “to” in “The thickness of the base seems to correspond: 127 μm”.

3.2. Raman Spectroscopy

I would suggest moving Figure 7 in the Supplementary Materials.

I suggest reformulating the final sentence of the paragraph as follows: “On the basis of the literature, the lower wavenumber region (200-1000 cm-1), mainly corresponds to skeletal vibration and ring deformation of the molecules [22], is the most relevant range for the identification of organic pigments [26]”.

Modify Figure 8 caption as follow “Raman spectra of the yellow, magenta and cyan dyes from Fujichrome Provia 400X Professional (RXP)”.

Modify the caption of Table 2 as follow: “Characteristic Raman vibrations possibly assignable to azomethine and indoaniline dyes [21, 24] for the spectra of yellow, magenta and cyan dyes from Ektachrome 160T Professional (EPT) (Figure 8) and from Fujichrome Provia 400X Professional (RXP) (Figure 9)”.

In Table 2 and Table 5, I would replace the assignment description “several structures” with “not specific”.

3.3. Thin-layer chromatography (TLC)

Merge Table 3 and Table 4.

Replace “of” with “by” in “The fact that two different solutions had to be used to elute each sample to achieve a proper separation of the dyes, immediately points out to films composed of different dyes”.

3.4. Fourier-Transform Infrared Spectroscopy (FTIR)

Correct the sentence “The dyes isolated from the preparative TLC were individually analysed…”

Modify the caption of Table 5 as follow “Characteristic IR vibrations possibly assignable to azomethine and indoaniline dyes [24, 27] for the spectra of dyes from Ektachrome 160T Professional (EPT) (Figure 10) and from Fujichrome Provia 400X Professional (RXP) (Figure 11)”.

3.5. High Performance Liquid Chromatography linked to Diode Array Detection (HPLC-DAD)

Move Table 6 in the Supplementary Materials.

Please merged Figure 12-13 and Figure 14-15. I would suggest using a letter/number/name to label the peak instead of the coloured circles.

Considering the high intensity of the peaks in 32-33 in range (Figure 14) they ca not be neglected. Some further comments? Cannot be related to some dyes by-products?

3.6. High Performance Liquid Chromatography coupled to Diode Array Detection and Mass Spectrometry (HPLC-DAD-MS) and Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS)

Why weren’t dyes extracted from RXP analyzed by HPLC-DAD-MS and LC-HRMS too?

Table 7: is it possible to report the comparison between experimental and calculated isotopic profiles not as screenshots?

Replace “more” with “most” in “The order of elution follows that one observed in the TLC analysis, confirming that M is the more polar dye followed by Y and C”.

The sentence “From the correspondent total ion chromatograms obtained in ESI positive and negative modes, it was extracted the m/z value associated to each peak” is not clear, please reformulate.

 

Why aren’t any chromatograms (TIC or EIC) reported? 

Author Response

Thank you a lot for your comments and suggestions. We think that the article has become much better with your review.

Author Response File: Author Response.docx

Reviewer 2 Report

This study is fascinating since only a few references are available on the presented topic. It provides a very well-written and dense introduction which helps the reader to understand the not-so-deeply investigated research topic. Furthermore, this work helps to find the gap regarding both the materials used in chromogenic reversal films and possible procedures to characterize them. I recommend publication after a minor revision.

Introduction

·       I suggest listing the techniques used in the order they were used to characterize the samples. This order should be followed also in their description in the materials and techniques section.

Materials and Techniques

·       The sentence “Both are 35 mm chromogenic reversal films are from the integrated dye couplers type.” should be revised;

·       I suggest inserting a photograph of the initial analyzed materials (maybe in the Supplementary file);

·       For each used technique, please explain on which type of samples did you perform the analyses, such as directly on the cross-section or on dyes’ extraction; It is not clear on which samples did you perform Raman spectroscopy; samples from cross-section or on dyes’ extraction?

·       Please remove the underline from paragraph 2.9

 

Results

·       I suggest putting together figures 3 and 4 and figures 5 and 6;

·       I suggest removing figure 7, it refers only to EPT and does not find an explanation in the text;

·       Be careful with the apex of cm-1 in the IR section;

·       Figures 8, 9, 10, 11, please add a legend to understand to what the spectra refer, maybe letters reported in the caption;

·       Figures 13 and 15, please add the unit of measurements of Absorbance, you could choose arbitrary units by removing the y value for example;

 

·       Please remove the underline from paragraph 3.6.

Comments for author File: Comments.docx

Author Response

Thank you a lot for your comments and suggestions. We think that the article has become much better with your review.

Author Response File: Author Response.docx