An Easy and Reliable Method for the Mitigation of Deuterated Chloroform Decomposition to Stabilise Susceptible NMR Samples

Round 1

Reviewer 1 Report

this work describes a method to wash the deuterated chloroform with concentrated Na2CO3 and subsequent desiccation with oven-dried Na2CO3, that works well for sensitive samples submitted to NMR analysis. I am supportive to publish this work, if the authors could compare this method with some previous solvent purification methods.

Author Response

Dear Reviewer 1 

Thank you for your suggestion to compare the described method with other methods. Such a comparison would be valuable, but, as stated in the draft, our searching has not found any clearly described other method nor any detailed description of the potential improvement as yet. Only rather short, abstract and imprecise general comments could be found, e.g.: “Use a small amount of activated, basic alumina.” “Dry with molecular sieve”  
In regard of the causes for varying NMR signal shifts or artefacts, i.e. acidity and the highly reactive phosgene, a fitting and simple method to tend to both causes was tested with clear results A comprehensive study of different potential methods was beyond the scope of our work. The comparison of this method with other reported procedures is hindered by the lack of detailed and reproducible publications, but then this creates an opportunity for further research. 

Kind regards
Jan Teipel (representing the team of authors) 

Reviewer 2 Report

Usage of CDCl3 for NMR is a relevant poorly disclosed topic. That’s why this manuscript can be interesting for readers.

The method developed is not a new one, but the authors show a good set of experimental examples. The methods are adequately described, and the results are clear.

Author Response

Dear Reviewer 2 

Thank you for your positive comments. 

Kind regards
Jan Teipel (representing the team of authors) 

Reviewer 3 Report

Very important topic for reliable analysis of NMR spectra in chloroform-d. Before acceptance I suggest a few small amendments.

1. It would be nice to assign signals in the presented spectra. To make the figures uncrowded I suggest to place such additionally labelled spectra in the Supplementary material.

2."defanged chloroform-d" in the title and text sounds a bit odd to a typical reader of chemical texts. Please, try to find a more popular expression. 

3. The presence of water signal (H2O) in Fig. 6 is visible at about 4.75 ppm. Please comment about the position of H2O in the spectrum and amount of water in the solvent and typical chemical shift of H2O and HOD in chloroform-d at about 1.60 and 1.55 ppm. The latter signal can appear as triplet. For example, see below:

Hoffman, R.E. 7402765254; Standardization of chemical shifts of TMS and solvent signals in NMR solvents (2006) Magnetic Resonance in Chemistry, 44 (6), pp. 606-616. Cited 89 times. DOI: 10.1002/mrc.1801 Gottlieb, H.E., Kotlyar, V., Nudelman, A. 7006197605;7005605504;7004349037; NMR chemical shifts of common laboratory solvents as trace impurities (1997) Journal of Organic Chemistry, 62 (21), pp. 7512-7515. Cited 2840 times. DOI: 10.1021/jo971176v

 

 

Author Response

Dear Reviewer3 

Thank you for your peer review and for your suggestions to improve the text.  

1.    This method to mitigate chloroform for NMR samples is of higher importance for non-targeted sample analysis where the sample’s NMR spectrum is seen as the sample’s fingerprint and evaluated as a whole (with the exemption of solvent signal regions). The molecular origin of individual signals is of lower importance for a reliable classification of the sample, but the reproducibility of a sample extract’s spectrum is essential. 
Just to offer some exemplary insight into the nature of our matrices some of the NMR signals were assigned to individual substances.
2.    In combination with the rest of the title, the figurative meaning of “defanged” should be quite clear to any reader of this publication, but in the spirit of scientific sober-mindedness the term was replaced with “mitigated” throughout the text. 
3.    The signal of residual water (H2O) at about 4.75 ppm, comparable to the chemical shift of HDO in D2O (4.79 ppm) or in CD3OD (4.87 ppm) hints at the existence of hydrogen bonds in the sample solution. A higher water content and the formation of clusters of water molecules in the solution can explain this observation. As described in literature minor traces of (isolated) water in chloroform will give a signal at 1.56 ppm. 
A somewhat higher water content in metabolomics NMR samples could result from insufficiently dried deuterated solvent but can also come from the original sample material: Thorough drying is often very effortful, a potential risk to the sample’s integrity and often not needed. 

Kind regards
Jan Teipel (representing the team of authors) 

Reviewer 4 Report

In this paper the authors described a new method to stabilize the deuterated chloroform and to avoid its decomposition (that normally occurs in presence of oxygen and water, leading to pH-shift and yielding to the presence of reaction products). The common stabilization methods have proven insufficient for sensitive NMR samples, so in this paper a new simple method is presented: the "defanged" chloroform-d was achieved by washing the deuterated chloroform with concentrated Na2CO3-solution and subsequent desiccation with oven-dried Na2CO3.

They presented three examples showing a high reproducibility (corresponding to less shifted NMR signals) in defanged than untreated chloroform. They showed also that a correct treatment with oven-dried Na2CO3 is important for the reduction of the water signal in the samples.

My main concern in this document is the discussion of the three examples. The data are not well presented and the figures are not well described both in the text (which makes data analysis complicated) and in the captions.

In my opinion they must improve the description of the obtained results.

Author Response

Dear Reviewer 4
 
Thank you for your peer review and for your suggestions to improve the text.  

To improve and numerically clarify the described results, we calculated (for some exemplary) signals their respective mean chemical shifts and the standard deviations, differentiating between samples made with untreated or with moderated chloroform. The numbers show as clearly as the figures that the reproducibility of signal positions is well enhanced if mitigated chloroform is used. 
By exemplarily assigning several signals to their source substances it should be clearer that these signals arise from relevant ingredients in the original samples. A full assignment of all signals to substances is beyond the scope of this work and is postponed until the publication of methods currently under development by the authors. Furthermore it is of lower importance since the relevance for non-targeted analysis is described in the introduction.  

Kind regards
Jan Teipel (representing the team of autors)