the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 03 Sep 2024
Method development and application for the analysis of chiral organic marker species in ice-cores
Abstract. Glaciers are valuable environmental archives that preserve organic compounds from atmospheric aerosols that can be used as marker species for their respective emission sources. Most environmental studies do not distinguish between the enantiomers of chiral compounds, although these compounds, mostly from biogenic sources, are very common in the atmosphere. We have developed a two-dimensional liquid chromatography (mLC-LC) method that allows the simultaneous determination of the chiral ratios of the monoterpene oxidation products cis-pinic acid and cis-pinonic acid in ice-core samples. The method combines a reversed-phase column in the first dimension and a chiral column in the second dimension in a simple instrumental setup with only one additional six-port valve. This novel method was successfully applied to selected ice-core samples from the Belukha glacier in the Siberian Altai spread over the period 1870–1970 CE. The chiral ratio of cis-pinic acid showed fluctuating values, while the chiral ratio of cis-pinonic acid remained more constant with an excess of the (–)-enantiomer.
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RC1: 'Comment on egusphere-2024-2243', Anonymous Referee #1, 13 Sep 2024
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This work presents a two-dimensional liquid chromatography (mLC-LC) method for the simultaneous determination of the chiral ratios of monoterpene oxidation products in ice-core samples. The method was applied to ice-core samples from the Belukha glacier, revealing fluctuating chiral ratios for cis-pinic acid and more stable ratios for cis-pinonic acid. The findings have the potential to impact related fields (e.g., aerosol chemistry), extending beyond the immediate scope of the ice-core analysis. While the work is both important and novel, some sections could be improved to enhance its quality.
The introduction is well-written and informative. However, since the main emphasis of the paper (as indicated by the title) is on method development, it would be helpful to include some rationale and an overview of the existing literature (at least a couple of lines) on the application of multiple heart-cutting 2D-LC. Why was this technique chosen over others? Some explanation on what “heart-cutting” 2D-LC should be provided as it is not a commonly used technique especially for AMT readers.The authors use water containing 2% ACN and 0.04% formic acid (A) and ACN containing 2% water (B). It would be helpful to provide a rationale for adding 2% ACN to phase A and 2% water to phase B, and /or citing appropriate literature (considering the journal's wider readership and the paper's focus on method development).
Line 176: The term "cut" in the sentence: "…and to rinse it completely before making the next cut" is unclear. Please rephrase for clarity.
Line 185: Replace “rudimentary” with “minimal.”
Section 3.3: Please include information on quality controls (QCs) and system suitability assessments.
Please clarify the number of injections in the following statement: "The instrumental repeatability was calculated as the standard deviation of multiple injections of a 100 ppb standard."
Why was the assessment done at 100 ppb (the upper limit of the reported linear dynamic range)? Typically, this is performed at low, medium, and high concentration levels (but below the max range, e.g. 70%).
Line 249: Add "whereas" before “E2 is formed by the oxidation of (-)-α-pinene.”
Line 250: Clarify what is meant by "the diagram" and make an appropriate reference. I assume the authors are referring to Figure 4 (as mentioned in the previous sentence).
Matrix effect is an important part of analytical method validation. Could the authors provide data or explain why this was not addressed in their study? While I realise that the authors rely on the high resolution and mass accuracy of the Orbitrap system, matrix effects, such as ion suppression, could be significant when using ESI, which is known to suffer from competitive ionisation. Was matrix-matched calibration used? This should be clarified in the validation section and addressed in the method application section as well.
What are the accuracy and precision (at a minimum of three concentration levels to ensure reliability across the analytical range. These levels generally include: low concentration (near the limit of quantification, or LOQ), medium concentration (close to the middle of the expected range) and high concentration (near the upper limit of the calibration curve). Again, considering the scope, the title of the paper and the type of samples, this is the minimum requirement in the method validation process to ensure that the analytical technique performs reliably across the specified concentration range.
Lastly, I suggest using absolute, rather than relative, intensity in Figure 3 to better demonstrate the true fluctuations in signal. This would provide a clearer picture of method robustness and any potential interferences.
Citation: https://doi.org/10.5194/egusphere-2024-2243-RC1 -
RC2: 'Comment on egusphere-2024-2243', Anonymous Referee #2, 23 Sep 2024
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This is a pretty straightforward paper. It describes an analytical methodology suitable for separating the two enantiomers (mirror image molecules) of a couple of organic compounds present in mid-latitude ice cores. A few values are shown as proof of concept. The method seems clever, simple and sound, as well as novel. The paper is written at a technical level that would be more suitable for an analytical chemistry journal, and I think a little extra explanation might be needed for a journal that is read by practitioners without that knowledge. I think that, in a journal that tries to link measurements to applications, it would be appropriate to write a little more about the motivation for this type of analysis, especially in the light of the results found (I will expand on this below, final comment).
Specific comments:
Line 72 “Enantiomerically pure (–)- and (+)-α-pinene (99%, optical purity ee: 97%) as well as cis-pinonic acid”. Please clarify, is the cis-pinonic also purchased as two separate enantiomers. I think not but from this sentence I am left unsure.
Section 2.2. Please give a little more detail about the ice core. What diameter was the core (10 cm?)? What depths were analysed, and in particular are the sections firn or solid ice (this is very important for the likelihood of contamination penetrating the sample, much more likely in firn).
Line 79-80. “The outer section was removed”. Please be more precise: “x mm was removed from the outside”. Did you do any tests to assure yourself that this was enough, eg by seeing if concentrations vary with distance from the edge of the sample?
Line 62 and elsewhere. Please explain at least once what the term “heart-cut” means as this will be unfamiliar to most readers, even to analytical chemists I think.
Line 112, 125 and surrounds. “Both dimensions were measured simultaneously”. I think I understand what is going on here but it could be explained more clearly (and perhaps I have misunderstood). If I understand it you are separating a lot of organic compounds in dimension 1, and just cut across to the second dimension for the two target compounds. This allows the enantiomers to appear within the chromatogram at times determined by their retention on both columns, embedded within other compounds whose timing depends only on their retention on one column. I’m not sure I see this as measuring both dimensions simultaneously, rather you ae embedding the results of the second dimension for 2 compounds within a mainly 1D separation. If I have indeed understood correctly, please give a clearer explanation of this.
Fig 3. What is “XIC” and what is the peak at 3 minutes?
Conclusions. It would add to the paper if you discussed at the end what your results imply about the application. I see two points. Firstly with the detection limit around the 1 ppb level, this method is appropriate for mid-latitude cores near to forests but it’s worth mentioning it is probably not yet applicable to polar ice cores with lower concentrations. Secondly, as I understand it you are hinting that the measurement of the enantiomers might allow discrimination of particular sources (eg tree species/forest type), or climate conditions. However it seems to me that the fact that you report that the enantiomeric ratios are different for two different oxidation products suggests that the ratio of the sources is not preserved, as does the observation that the predominance of the (-) enantiomer is not what you’d expect for a boreal forest. This is disappointing, and I think it would be appropriate to admit that this makes it difficult to see how the enantiomeric ratios can be used to differentiate sources, even if it suggests other lines of research. In other words, while this paper is an excellent analytical achievement, I think it should admit that its application looks very difficult indeed.
Citation: https://doi.org/10.5194/egusphere-2024-2243-RC2
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