Reply on RC1

The manuscript by Diao et al. investigates different fractionation processes caused by Hexchange with organic material, sublimation and evaporation processes during cryogenic vacuum distillation of plant samples. The manuscript is certainly of high interest to scientists working with CVD and provides valuable insights into different fractionation processes. However, I feel that the study has certain shortcomings, which prevent the certainty with which some conclusions were made. I also feel that the description of the experiments + results and discussion could be improved to enhance the clarity of the manuscript. Please find my major and minor comments below.


244-247).
(2) Drying procedure of sample material: why did you dry your material only at 60°C and for 24h? I know for organic materials it is common to use 60-70°C. However, I feel that for such an experiment a completely dry sample (105°C) would have been of immense importance. At least it should have been dried for 48h. This has to be addressed somehow and could potentially compromise your results. Same issue in L. 128.
Response: Thank you for pointing this out. In our study, the drying procedure of sample material was only for experiment 1. Because the dried samples in the experiment 2 were rehydrated with excess of water for isotopic equilibration and no dried materials were used in the experiment 3 and 4.
For the stem and twig pieces, the original materials were first dried at 60°C for 24 h in order to cut them into pieces and then for weighing. Then, 200 mg of pieces were weighed in Exetainers and dried again at 60°C for 24 h. In total, the sample were actually dried for 48 h as suggested by the reviewer. For the powdered materials, they have already been dried and stored in a dry environment prior to the experiment. Those samples were used as isotope reference standards for δ 2 H analysis in our laboratory (Schuler et al. 2022). So, the powdered materials were further dried once in the Exetainers at 60°C for 24 h. We added some sentences to clarified this. See lines: 106-107; 112; 118; 122-126. We also would like to note that it is very difficult to have completely dry samples, even if at 105°C for 48 h. That's because moisture in the air will be absorbed within seconds by the dry samples when the samples are taken out from the oven and when opening the cap for injecting the reference water.
To address potential problems regarding our sample drying procedure, we conducted a test using ca. 200 mg stem pieces, stem powder and caffeine at two different drying procedures (60°C, 48 h; 105°C, 48 h The results show that all the tested materials were dried to a constant weight after 36 h and 12 h at 60°C and 105°C, respectively. However, more moisture was removed at 105°C, especially for twig pieces and stem powder. Noticeably, after opening the cap for 5 s, the samples which were dried at 105°C absorbed more lab water vapour than the samples which were dried at 60°C. These results suggest that a complete drying is very difficult, regardless of whether the samples were dried at 60°C or 105°C. However, the difference of 1-2 % in removed moisture after reabsorption between 60°C and 105°C should not affect the isotopic results of our study, because strongly depleted reference water was used to amplify the differences. It should also be noted, that the higher uncertainty at lower amounts of water is observed across all our experiments, again showing that the remaining moisture in the dried material is not a major driver of our overall results. We have added this table to the supplement, see the new Table S2. Corresponding texts were added at lines: 132-139; 255-264. (3) Over-/undersaturation of sample material: So if some water amounts were not able to fully saturate the material and others oversaturated the material, I would be really careful with the conclusion drawn from this experiment, as it does not reflect "real" plant samples. If relative water content is not an issue, why did you not choose different weights of your samples material to avoid over-/undersaturation?
Response: First, the main objective of Experiment 1 was to investigate the phenomena of H-exchange of different plant material and compounds across a range of water to biomass ratios. It was not intended to reflect a natural gradient of relative water content. Second, given that the isotopic variation along the gradient were similar across material and reference water (without any organics), led us conclude that AWA rather than RWC is important. We thus designed experiment 2 to demonstrate this point. We added text indicating that we intended to generate a range of water/biomass ratios (Line 120-121).
As a general consideration, we would also like the to mention that controlled experiments, as we did, always have some advantages (like testing for certain effects), but also disadvantages (like not necessarily being representative for "real" samples).
(4) In general, there are a lot of assumptions in the material and methods sections, such as "Thus, by the end of the rehydration, the isotope ratios of water in the small stem segments are assumed equal to the isotope ratio of the reference water after rehydration (δref after rehyd) and not to be equal to the original reference water (δref)." (L. 137-139). Can you be sure about this? This should be considered in the discussion.
Response: Good point. Indeed, we cannot be 100% sure whether the exchangeable H in the different material was in full isotopic equilibrium with the water, as we can't determine the isotope ratio of the exchangeable H in the material after rehydration. However, at least some percentage of the exchangeable H will exchange with the water under the chosen experimental conditions (25°C), particularly those that are not linked via hydrogen bridges (Sepall and Mason, 1961). As indicated by experiment 1, the input of hydrogen derived from organic material on the reference water can be significant at lower water amounts. We thus note that the original isotope ratio of the reference water has changed and that the isotope ratio of the reference water after exchange reflect a better reference. Several sentences have been added at the end of the second paragraph of section 3.2 in the Discussion to address this issue (Line 296-303).
(5) Experiment 3: I am afraid I don't really get the whole experiment. From what I understand you were only interested in the effects after the water has been extracted from the sample, thus the freezing in liquid nitrogen. But why do you then write "before the extraction started"? I guess you left the water in the liquid nitrogen for a certain amount to simulate an extraction? Why not also freeze the reference water at -20°C and extract it the way as in experiments 1 and 2? This would also allow a statement of the effect of heating the water and potential evaporation effects before freezing the water. There is certainly some clarification needed for this experiment.
Response: Sorry for causing this misunderstanding. In experiment 3, the reference water was also frozen, simply in the U-shaped water collection tube with liquid nitrogen, not in the Exetainer in a freezer at -20°C (as in Experiment 1 and 2). So, before the extraction started the water was frozen, then sample tubes and the collection tubes were put in the 80°C water bath and liquid nitrogen, respectively, and vacuum applied. That's why we wrote "before the extraction started". We have revised section 2.3 to clarify those unclear descriptions (Line 165-167).
With respect to "freeze the reference water at -20°C and extract it the way as in experiments 1 and 2", we have already done that experiment. This is actually our "pure reference water extraction" in Fig 1b. A description of this can be found in the last paragraph of section 2.1: "As a control, the experiment was repeated without any material by adding only the range of reference water into the vial." (Line 129-130). So, in experiment 3, we went in-depth to see what happened during the sublimation of the frozen extracted water.
(6) As four experiments were conducted, it is sometimes really confusing for the reader to follow the argumentation, as you always have to go back to the methods to see, which experiment exactly the authors are talking about. Potentially, Fig. S1 could be moved into the manuscript, but in a clearer manner with a clearer description.

Response:
We have thoroughly revised Fig. S1 to make the individual experiment more understandable. As suggested, we moved Fig. S1 into the manuscript, see the new Fig. 1. (7) Figure 1-3: There are some undiscussed effects in Figure 1 and 2: Fig. 1: why do powder materials drop below the reference line at high water amounts? This remains unexplained

Response:
We have discussed the difference in Δ 2 H between pieces and powdered materials when AWA > 600 μl in third paragraph of section 3.1 (Line 247-255). A possible explanation is that the isotopic compositions in the exchangeable H of pieces and powdered samples were different. Fig. 2: for H, your dH is negative for water samples larger than 400 microl, for 18O, they are still clearly above the reference line. This should certainly be discussed.

Response:
The difference between the Δ and the reference line might be explained by analytical uncertainties in δ ref , causing offsets in Δ to the reference line. We therefore added the standard deviation of the reference line in the original Fig. 1 and Fig. 3 to express this uncertainty (now they are Fig. 2 and Fig.4). In these figures, all the δ values were referenced against one single δ ref value (an average value from repeated measurements of the reference water). The standard deviation of the reference line was not added in the original Fig. 2, because each point in this figure has its own reference value (i.e., δ ref after rehyd ); the standard deviation of the reference line was not added in the original Fig. 4 because δ CVD_ave was taken as a reference. Fig. 3: for H in your sublimation and evaporation test, dH is steadily decreasing with absolute water amount. You argue that this is analytical uncertainty, however it is a quite clear pattern, which remains undiscussed. Also, the fit for dH in the sublimation experiment is somewhat off and not well representative of the data.
Response: Sorry for causing this misunderstanding. We agree that the Δ 2 H is steadily decreasing with absolute water amount and that this pattern is very clear. The analytical uncertainty argument was made on the negative values in Δ 2 H at AWA > 600 μl, not on the overall decreasing pattern. See the text: "We suppose that the negative Δ 2 H and Δ 18 O values for the AWA > 600 μl were purely caused by the analytical uncertainty, because no incomplete extraction could occur given that the reference water was added directly into the collection tube." (Line 333-335) To be consistent, we used the same model fits for the results from all the experiments. The reason for the fit for Δ 2 H in the sublimation experiment not well representative of the data is probably that the Δ 2 H of samples at 100 and 200 μl were somewhat shifted up a little bit compared with the fitted line.
(8) Recommendation to extract more than 600 microl: From the results, I cannot clearly see, why the authors recommend at least 600 microl of water for extraction. In Figure 2 you clearly see that at 600 microl there is still quite a substantial offset for 2H and 18O, in Fig. 3 this is also the case for the evaporation experiment and 2H. In Fig. 4 there is a negative offset at 600 microl for 18O. This should be discussed in more detail.
Response: We agree that the exact level of 600 μl is somewhat arbitrary, but it was chosen because (i) the discrepancy among Δ values at AWA > 600 μl were much smaller than those < 600 μl; (ii) despite there are still offsets when AWA > 600 μl, the offsets were relatively small and within the range of systematic error of the isotope ratio of the reference water (i.e., standard deviations are now given as grey bars). Therefore, extracting a minimum amount of 600 μl of water could reduce large isotopic enrichments. We have added some sentences to discuss this in more detail: See lines: 231-232; 430-431.
(9) Plant material: almost all experiments were only conducted on Larix decidua. This should be included in the discussion, as the results could be completely different for other woody species and especially for herbaceous plants.