From the journal Environmental Science: Atmospheres Peer review history

25-Apr-2021

Dear Dr Styler:

Manuscript ID: EA-ART-02-2021-000011
TITLE: Unraveling the complexity of atmospheric brown carbon produced by smoldering boreal peat using size-exclusion chromatography with selective mobile phases

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Dr Nønne Prisle
Associate Editor, Environmental Sciences: Atmospheres

************

Reviewer 1

This is an important paper that probes the limitations of size exclusion chromatography (SEC) for the analysis of molecular size distribution in brown carbon (BrC) from biomass burning. Studies relying on SEC to classify BrC by molecular size started to appear a few years ago, and in my opinion, they suffered from limitations of using calibration standards that have very different intermolecular interactions with the matrix than natural samples do. This work shows that SEC data from the same sample depends on the eluent, to the extent that very different molecular sizes are measured even if calibration with typical SEC standards is done under the same conditions. The authors proceed to demonstrate that increasing the fraction of acetonitrile in the eluent breaks down BrC supramolecular aggregates as well as has an effect on interaction of eluents with the stationary phase. This result is important for people who investigate the molecular vs aggerate nature of BrC chromophores. I would publish this paper with minimal changes outlined below.

MINOR COMMENTS:
1). The paper would benefit from a stronger discussion of how these results affect the interpretation of previous studies. Some people believe that BrC chromophores are mostly molecular, and some people believe (based on previous SEC studies) that they are dominated by high-molecular weight species. This study shows that both types of chromophores contribute. It would be good to re-examine conclusions achieved by all previous SEC studies in view of these new results.
2). The authors make a fra-reaching conclusion that BrC of different age behave differently, however, I do not think data in this study fully support this. The authors would need to compare the behavior of BrC from the SAME source before and after aging. As far as I understand, what the authors do instead is comparing results of field measurements (that come from many sources) and peat combustion (that is a rather unusual source for the area in which authors reside). Maybe I am missing something but I would not emphasize fresh vs aged comparison, e.g., remove this from the abstract. I would carry out a separate dedicated study on the effect of aging and publish it separately.
3). I would suggest moving the AF4 segment from the SI section to the main text.
4). “These results suggest that MeOH is less effective than ACN at disrupting hydrophobic interactions between the sample and the column matrix.” – there is probably literature on this to support this statement, likely in biochemical journals looking at unfolding of proteins.


TECHNICAL CORRECTIONS:
Line numbers: I would have appreciated including those in the draft. Fortunately, the paper is very well written, so I do not have many technical corrections.
Figures in the text and SI: some figures are a little granny, especially S1
Abstract: SEC used without defining it in the last sentence
Ref 1, 21: missing year, volume and pages
Ref 35, 71: title is in all caps
Ref 38: year missing
Figure S1: would recommend adding lines and equations used for calibration calculations

Reviewer 2

This paper investigates the composition of brown carbon species produced from smoldering boreal peat. It developed a specific method and applied it successful to achieve this goal. The paper is generally well written and presents a good contribution to our understanding of BrC composition and its link with the light absorption properties. This reviewer recommends its publication, and only has a couple of minor comments:
(1) The light absorption properties are not measured by common techniques like UV-Vis spectrometry, any more comments on their differences?
(2) Any more molecular insights to the composition of BrC by using this method rather than only vaguely saying groups/types of the BrC species?

Dear Dr. Prisle:

On behalf of my co-authors, thank you for the opportunity to submit revisions to our manuscript. I have included here our response to the reviewers' reports.

Sincerely,
Sarah Styler

_____________

“Unraveling the complexity of atmospheric brown carbon produced by smoldering boreal peat using size-exclusion chromatography with selective mobile phases”, by Lyu et al. (EA-ART-02-2021-000011)— response to reviewers

We thank the anonymous reviewers for their insightful comments and suggestions. Here, we address each comment in turn. Please find below the reviewer comments numbered and in bold/italics; changes to the manuscript that have been made in response to the reviewer comments are summarized in bold blue text, both here and in the marked-up resubmission.

Reviewer 1

1 “This is an important paper that probes the limitations of size exclusion chromatography (SEC) for the analysis of molecular size distribution in brown carbon (BrC) from biomass burning. Studies relying on SEC to classify BrC by molecular size started to appear a few years ago, and in my opinion, they suffered from limitations of using calibration standards that have very different intermolecular interactions with the matrix than natural samples do. This work shows that SEC data from the same sample depends on the eluent, to the extent that very different molecular sizes are measured even if calibration with typical SEC standards is done under the same conditions. The authors proceed to demonstrate that increasing the fraction of acetonitrile in the eluent breaks down BrC supramolecular aggregates as well as has an effect on interaction of eluents with the stationary phase. This result is important for people who investigate the molecular vs aggerate nature of BrC chromophores.”

Thank you!

2 “The paper would benefit from a stronger discussion of how these results affect the interpretation of previous studies. Some people believe that BrC chromophores are mostly molecular, and some people believe (based on previous SEC studies) that they are dominated by high-molecular weight species. This study shows that both types of chromophores contribute. It would be good to re-examine conclusions achieved by all previous SEC studies in view of these new results.”

We have amended our conclusions section to include a more detailed discussion of the implications of our work for SEC analysis of BrC:


“This study represents the first systematic investigation of the challenges and benefits associated with the application of HPLC-SEC-PDA to the characterization of atmospheric brown carbon (BrC), an important class of light-absorbing PM and the dominant contributor to the absorption profile of wildfire PM. In the following paragraphs, we highlight the implications of our results for SEC analysis of atmospheric samples and provide recommendations for future work in this area.

A major conclusion of this study is that careful consideration of solvent–analyte interactions is crucial when identifying appropriate extraction and chromatographic/spectroscopic analysis conditions for natural samples. Previous work has shown that the composition of PM extracts can be influenced by reactions of specific organic functional groups with sample extraction solvents (e.g., MeOH).71 Here, we show that organic solvents can influence BrC properties even without reacting directly with BrC components: in particular, we show that ACN not only mitigates hydrophobic interactions between BrC and the SEC column matrix but also disrupts metastable BrC aggregates, thereby leading to changes in measured BrC size distributions. This aggregation and dissociation behaviour could potentially contribute to discrepancies between MW values inferred from SEC and those determined using mass spectrometry for the same sample.

Importantly, and more broadly, our findings imply a need for caution when using chromatographic elution behaviour (e.g., in reversed-phase HPLC) to make inferences regarding the hydrophobicity and other properties of BrC, as the relationship between retention time and mobile phase identity for a given BrC component may reflect both changes in its distribution constant and changes in its intrinsic properties. For example, increases in ACN mobile phase content (e.g., in gradient methods) could potentially lead not only to changes in partitioning behaviour but also to dissociation of BrC aggregates, with concomitant indirect changes in retention behaviour.

Successful application of SEC to MW estimations of complex mixtures is a challenging task, as it requires both selection of calibration standards with comparable chemical and structural properties to the analytes of interest and careful consideration of biases resulting from differences in the magnitudes of secondary effects experienced by mixture components compared to the chosen calibrant suite. For example, previous studies applying SEC to analysis of atmospheric PM have employed both commercial macromolecules and atmospherically relevant small molecules (e.g., phenols and aromatic acids) for MW calibration. However, given that we would expect these calibrants to be differently susceptible to secondary effects than BrC, this strategy has the potential to lead to dramatic changes in estimated MWs with changing solvent composition. In the case of humic substances, these issues have occupied analytical chemists for decades. In the case of fresh BrC, which we show here is made up of components with a wide range of sizes, polarities, and susceptibility to conformational changes—and therefore subject to different secondary effects—achieving size-based separation of all BrC components at any single mobile phase composition, no matter how optimized, is likely an impossible task. This challenge is especially important in cases where SEC is coupled with complementary techniques to investigate the chemical properties of specific analyte size fractions, as a given elution volume could potentially contain contributions not only from “ideal” analytes (i.e., those for which secondary interactions have been minimized) but also from many other analyte classes (e.g., higher-MW but more hydrophobic, smaller-MW but susceptible to intramolecular electrostatic repulsions).”

3 “The authors make a fra-reaching conclusion that BrC of different age behave differently, however, I do not think data in this study fully support this. The authors would need to compare the behavior of BrC from the SAME source before and after aging. As far as I understand, what the authors do instead is comparing results of field measurements (that come from many sources) and peat combustion (that is a rather unusual source for the area in which authors reside). Maybe I am missing something but I would not emphasize fresh vs aged comparison, e.g., remove this from the abstract. I would carry out a separate dedicated study on the effect of aging and publish it separately.”

Thank you for these useful comments. We also realize the importance and necessity of comparing BrC from the same source before and after aging. In fact, we are currently preparing a companion manuscript that reports our investigations of the photolysis of different BrC samples (as aqueous extracts), including BrC produced by the combustion of boreal peat sampled from different depths and with different moisture contents and BrC produced by the combustion of spruce foliage.

Interestingly, we find significant sample-to-sample variation in the evolution of the size-dependent light-absorbing properties (as analyzed using SEC-PDA) of BrC with photolytic aging, and that the high-MW and low-MW fractions behave differently upon illumination. We discuss these results in detail in this second manuscript, which we will be submitting to the Mario Molina Memorial Special Issue of ACS Earth and Space Chemistry later this spring.

In response to these comments, we have deleted the following text from the abstract:

“the role of atmospheric aging in mediating BrC properties”

In addition, we have edited the abstract to make it clear that results for atmospherically aged wildfire BrC were obtained from previous studies, rather than from this study:

“Unlike atmospherically aged wildfire BrC, which has previously been shown to resemble terrestrial humic substances in both its absorption profile and its retention behaviour, the fresh BrC sample studied here”

We have also edited the text of the manuscript to reflect that a) multiple explanations could exist for the differences observed between our samples and ambient BrC samples studied in previous experiments and b) definitive conclusions regarding these differences would require examination of results from aging experiments:

“These differences could reflect fundamental differences in sample types: whereas our BrC sample is solely from peat combustion, the BrC samples studied by Di Lorenzo et al. were collected from a variety of locations, and would therefore be expected to include contributions from multiple fuel types—and, in some cases, urban and industrial emissions. These differences could also reflect changes in BrC properties during atmospheric transport: for example, previous aircraft and laboratory studies of water uptake by biomass burning PM have shown an increase in particle hygroscopicity with increasing particle oxidation. Additional evidence for the role of atmospheric aging is provided in the Di Lorenzo et al. study itself, which showed that the contribution of high-MW chromophores to overall BrC absorption increased with plume age; therein, the authors attributed this change to the in-plume (photo)oxidation and/or oligomerization of low-MW species. Since our BrC sample was collected immediately after its release from the combustion source, we would expect it to be less oxidized, more hydrophobic, and less depleted in low-MW chromophores than even the “freshest” BrC sample in the aforementioned work, all of which is consistent with our observations. To fully understand these differences, however, investigation of the aging behaviour of our specific BrC sample would be required.”

4 “I would suggest moving the AF4 segment from the SI section to the main text.”

We have moved the AF4 methods section and Figure S8 (now Figure 2) to the main text; the added section text and figure caption are highlighted in blue bold text in the revised submission.

5 “These results suggest that MeOH is less effective than ACN at disrupting hydrophobic interactions between the sample and the column matrix.” – there is probably literature on this to support this statement, likely in biochemical journals looking at unfolding of proteins.”

We have added the following text:

“These results, which are unsurprising given the relative elution strengths of these solvents in reversed-phase partition chromatography (i.e., ACN > MeOH), suggest that MeOH is less effective than ACN at disrupting hydrophobic interactions between the sample and the column matrix.”

6 “Line numbers: I would have appreciated including those in the draft. Fortunately, the paper is very well written, so I do not have many technical corrections.”

We have included line numbers in the revised submission.

7 Figures in the text and SI: some figures are a little granny, especially S1
10 Figure S1: would recommend adding lines and equations used for calibration calculations

We have added linear regression fits to Figure S1 and have improved its resolution. To avoid cluttering the legend in Figure S1, we have placed calibration curve equations together with SRHA MW estimations in Table S1.

8 Abstract: SEC used without defining it in the last sentence

We have edited the abstract as follows:

“Here, we use size-exclusion chromatography (SEC) coupled with photodiode array detection to characterize BrC collected from the controlled combustion of boreal peat.”

9 Ref 1, 21: missing year, volume and pages; Ref 35, 71: title is in all caps; Ref 38: year missing

We have made these corrections in the revised submission.


Reviewer 2

1 “This paper investigates the composition of brown carbon species produced from smoldering boreal peat. It developed a specific method and applied it successfully to achieve this goal. The paper is generally well written and presents a good contribution to our understanding of BrC composition and its link with the light absorption properties. This reviewer recommends its publication, and only has a couple of minor comments”

Thank you!

2 “The light absorption properties are not measured by common techniques like UV-Vis spectrometry, any more comments on their differences? ”

In this study, we used a photodiode array (PDA) detector coupled to a SEC column to obtain the UV-Vis absorption profile (200–600 nm) of the SEC eluent; this approach provided us the 2D absorption density plots shown in the manuscript. Because of the compositional complexity and limited volume (1.5 mL per sample) of our BrC extracts, we did not obtain conventional UV-Vis spectra for our samples. However, the 2D absorption density plots are sufficient to reveal sample UV-Vis absorption properties, and provide additional information regarding chromophore characteristics (e.g., hydrophobicity, conformation) that is not accessible for non-size-separated samples.

For example, whereas conventional UV-Vis spectra provide total wavelength-dependent sample absorption, these 2D plots enable us to investigate the light absorption at specific wavelengths for different molecular size fractions, and in particular to show that BrC consists of two characteristic groups: the first, humic-like, group exhibits featureless absorption that sharply decreases from UV to visible wavelengths; the second group, either smaller in size or more subject to hydrophobic interactions, showed structured absorption (i.e., centred at specific wavelengths). These differences, however, could be masked or partially obscured in the overall UV-Vis spectrum.

3 “Any more molecular insights to the composition of BrC by using this method rather than only vaguely saying groups/types of the BrC species?”

In this study, we report the light-absorbing properties of BrC as a function of molecular size. We find that our BrC sample contains hydrophobic components, is subject to disaggregation under certain mobile phase conditions, undergoes conformational changes with changes in mobile phase ionic strength, and exhibits “tyrosine-like” fluorescence.

Molecular-level characterization of BrC is subject to its own limitations and artifacts, which we describe in the introduction (e.g., multiple charging effects, fragmentation, co-elution effects). Although this characterization strategy is beyond the scope of the current study, to more clearly highlight work in this area, we have amended the introduction to include insights into BrC composition obtained using high-resolution mass spectrometry (HRMS):

“Molecular-level BrC characterization is usually accomplished via offline analysis of PM extracts using high-performance liquid chromatography (HPLC) coupled with photodiode array (PDA) and high-resolution mass spectrometric (HRMS) detection; however, summing the contribution of chromophores identified by MS (e.g., N-containing aromatic compounds, aromatic carbonyls, and aromatic carboxylic acids) typically cannot account for all BrC absorption.”

“In addition, the analysis of the high molecular-weight (MW) component of BrC (often referred to as “humic-like substances”, or HULIS), which consists of macromolecular aggregates composed of highly conjugated aromatic structures with polar functional groups containing oxygen and nitrogen (e.g., carboxyl and nitro groups), may be complicated by fragmentation in the MS and by multiple charging effects.”

07-Jun-2021

Dear Dr Styler:

Manuscript ID: EA-ART-02-2021-000011.R1
TITLE: Unraveling the complexity of atmospheric brown carbon produced by smoldering boreal peat using size-exclusion chromatography with selective mobile phases

Thank you for submitting your revised manuscript to Environmental Science: Atmospheres. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Dr Nønne Prisle
Associate Editor, Environmental Sciences: Atmospheres

Reviewer 1

I commend the authors for their careful approach to addressing all reviewers’ comments. The paper is ready for publication in my opinion. I am going to select “publish as is” and list some minor corrections below that authors can ignore, they are not too important to address.
Page 9: “1690 Da - 78.4 kDa” -> “1.7-78 kDa. I would similarly replace Da by kDa in the SI section.
Page 10: “ranged from 1000 to > 10000 Da” -> “ranged from 1 to > 10 kDa”
Retention times listed in tables S1 and S2 appear to have more significant digits than one would expect based on the method precision.