Average value of Area1600 cm-1/Area1777 With this quantitative determination method in hand, we then used it to determine the degradation percentage in the samples from the methanolysis, and to extrapolate from it the average polymerization degree all along the reaction progress. Data are presented in Table , in comparison with the values calculated from the SEC analysis. Based on our calibration curve, we could first observe that the first two points (t = 0 and 2 min) fall outside the calibration range. The depolymerization percentage at 2 min being 0.39 % according to the SEC analysis, this clearly shows that FT-IR is not suitable for such low degradation rate, unlike SEC. Then, from 5 min to 120 min, the average polymerization degree ranged from nearly 11 to 2.5, values that are close to those derived from the SEC analysis (14 to 3.6 respectively), despite the latter being only estimates as stated before. We should point out, however, that as our calibration curve was made only with a mixture of BPA/PBPAC, it can only serve as an approximation for the analysis of methanolysis, which also generates products with methyl carbonate-bisphenol A chain ends, which are not taken into account in the analysis of peak ratios detected in FT-IR. That said, the correlation between the two methods shows that these approximations are actually quite small compared with the overall monitoring of the degree of depolymerization, in particular as long as we place ourselves in percentages of degradation higher than the LOD of the two approaches. As a consequence, we may conclude that it is therefore be possible to use FT-IR to obtain a somewhat quantitative idea of the PBPAC depolymerization, with a minimal detection limit of 10 %. In such sense, it could represent a good alternative/complement to SEC as, unlike FT-IR, the latter is fairly complex and expensive to perform, requires important post-processes depending on the reaction mixture, is quite solvent-intensive, most importantly, quite low throughput. With only a few seconds per spot, the need for a few µL droplets of sample and the possibility of using it in a 96-well plate format compatible with a liquid handler, FT-IR could be much more suitable for screening campaigns where large collections of catalysts and sets of reaction conditions need to be rapidly evaluated. Especially, for biocatalyst research for example, given the difficulty of finding enzymes or strains capable of acting on this family of polymers, this type of approach seems to be essential for exploring biodiversity to the maximum, or optimizing the catalytic capacity of species by rational design, for example. As a proof of concept, we have recently succeeded in fully automating sample deposition on FT-IR targets with a Biomek i7 robot (Beckman Coulter), thanks in particular to the development of an adapter making the target compatible deck of the latter (Figure ). Nevertheless, it is not advisable to rely solely on FT-IR when the depolymerization rate does not reach 10 %, and this must be considered in advance of test campaigns, in anticipation of the yields expected for the catalysts and conditions tested. Complementary to FT-IR, we have also analyzed our PBPAC depolymerization reaction by 1 H NMR to assess its efficiency and compare it to the one of the former, particularly in terms of its minimum detection limit, as well as the different levels of information it can potentially offer. The resulting spectra for each time point are shown in Figure , with a focus on the 1-7.7 ppm chemical shift region (full spectra are available in figures S11-16). First of all, in the region where the protons of the two methyl groups are located, we can observe depolymerization through the progressive decrease of the singlet at 1.68 ppm, until its virtual disappearance at 120 min. In addition, from 5 min of reaction, the appearance of a new singlet at 1.64 ppm can be observed. For the rest of the study, we used this region as a proton-equivalent reference for the integrations. We therefore set the integration of the sum of peaks at 6 (i.e. 2 methyl groups with 3 protons each). Notably, the integration for these two peaks at 5 min was respectively 1.2 and 4.6 proton equivalents. This corresponds to 23.4 % of methyl groups that are not located inside the polymer chain, which is within the expected range according to previously calculated yield, as for 10 % degradation, we can expect 20 % of these protons since a cleaved bond produces 2 chain ends. At 15 min, 5 new singlets are observed at 1.62 ppm, 1.64 ppm, 1.65 ppm, 1.66 ppm and 1.67 ppm. Based on the previously determined 1 H NMR spectra, the singlets at 1.62 ppm and 1.68 ppm could be assigned respectively to the methyl protons of BPA and the PBPAC repeating unit. In 2008, were able to use 13 C NMR to observe the carbons assigned to the methyl at the end of the chain by the appearance of slightly changed chemical shift signals from the methyl in the repeated unit. This is explained by the fact that the end unit does not contain a carbonate bond on both sides, unlike the repeated unit, leading a slightly different shielding effect. Similarly, when applied to our samples, the appearance of singlets at 1.64, 1.65, 1.66 and 1.67 ppm can be assigned to the methyl protons of the different end groups of the reaction intermediates, identified by MALDI (Table ), although each individual peak cannot be precisely attributed. A second revealing region is of course that of the aromatic protons, between 6.5 to 7.5 ppm. The first changes in this region can be seen after 5 min of degradation, with the appearance of new signals as well as the decrease in intensity of the protons of the repeating unit, which continues until 120 min. The chemical shifts of these new peaks were between 6.67 and 6.75 ppm and between 7.03 and 7.20 ppm. Unfortunately, the overlapping of the signals makes it impossible to determine the multiplicity, neither the exact integration of each signal. However, keeping the same reference as for the methyl protons, the sum of the integrations of all the signals in the aromatic region is equal to 8 equivalent protons, as expected. Also, as in the previous case, these new signals can be attributed to the aromatic protons at the end of the chain of intermediates, even though they do not help to know how many new species are effectively produced. Finally, the region around 3.8 ppm corresponds to the methyl protons of the various methyl carbonates generated from the methanolysis. We can thus see 2 singlets with almost identical chemical shifts (3.892 and 3.894 ppm respectively). Given the population of methyl carbonate chain ends detected by MALDI-TOF (series E and F peaks), it is possible that at least one of these two singlets corresponds to the latter. Note that it is very likely that both populations have the same chemical shift for these protons, especially as the number of polymeric units is large, as the environment is very similar for each end of the chain. On the other hand, it could be that the second singlet corresponds to the methyl protons of the methyl carbonate attached to a released BPA molecule, which could have a slightly different chemical shift, although it is not really possible to confirm this from these results alone. Finally, note that the triplet at 3.76 ppm corresponds to THF remaining after evaporation, as can be seen from t = 0 in very small proportions. As a main conclusion to this analysis, we can first confirm once again that depolymerization has indeed taken place, as observed with the other analytical methods, and that 1 H NMR offers several markers to track it correctly. On the other hand, since most of the signals here come out of the same zone, it's rather difficult to distinguish between the degradation products, let alone identify each of them precisely. As with the other techniques, here too we can calculate the minimum detection limit. If we refer to the percentage of degradation determined in SEC, and taking into account the fact that the first new signals are only distinguishable in 1 H NMR from 5 min onwards, this would amount to a limit of around 8 % depolymerization. This makes 1 H NMR a slightly more sensitive approach than FT-IR. However, in addition to the limitation sported regarding the determination of the intermediate products, NMR is like SEC, being quite limited by certain technical constraints (low sample throughput, expensive deuterated solvents, etc.), As such, it would only be an interesting alternative or complement if we were to use a more advanced analysis of the spectra, perhaps by carrying out long-distance coupling experiments to better identify and characterize the species present. In this case, it should be used at the end of the analysis to confirm certain by-products that are difficult to identify, or to validate results obtained with high-throughput approaches, for example. Finally, as for FT-IR, NMR is also is highly dependent on the purity of the analyte, requiring an important sample post-processing if used on complex degradation media such as fermentations. Finally, as already mentioned, BPA release into the reaction medium can be quantified easily by HPLC, in order to confirm the actual yield of conversion, but also to assess if the degradation appears to be complete or stops at a certain degree of polymerization. The chromatograms obtained from the different samples are shown in Figure . Unlike the other techniques used previously, the first changes are detected only after 15 minutes of reaction, with two peaks with respective retention times of 6.59 and 7.67 min. However, if the depolymerization mechanism does indeed take place endo as we hypothesized, this seems quite logical as the chances of breaking a random bond within the polymer chain, producing oligomers, are much higher than those of hydrolysis of the polymer ends which would release BPA. Concerning the product attribution, the first peak appears to be BPA monomer, in accordance with the first measurement done and the standard chemicals. However, the second peak could not be identified with certainty, notably due to the absence of oligomer standards in our possession. Nonetheless, its retention time being higher, significantly longer than that of BPA, implying a lower polarity of the molecule compared to BPA. Consequently, while it could correspond to the more hydrophobic methyl carbonate derivative of BPA, a product in which one of the chain ends has not undergone double transesterification as identified by MALDI-TOF. Using our calibration curve, we could however estimate the concentration of BPA to be 0.22 mM after 15 min, which corresponds to a molar conversion yield of 1.1 % of polymer to pure monomer. The fact that no monomers could be detected before this time also leads us to be able to determine a minimum detection limit for polymer degradation of 20 %, by correlation with the SEC results. Although quite high in comparison with other limits measured before, this is again quite relevant under our conditions, since HPLC here cannot allow us to detect anything other than total depolymerization of the polymer. In consideration of the extent of degradation that could be quantified by HPLC, it is notable that the maximum concentration of BPA recorded after two hours of reaction was 3.9 mM, which corresponds to a final molar conversion of 19 ± 4 % of PBPAC to pure BPA. As a result, without additional standards, that can be particularly hard to obtain for some polymers, including PBPAC, HPLC analysis provides only partial results on depolymerisation. Nevertheless, this approach remains highly accurate for quantifying the release of BPA into the medium, with a detection limit of 6 µM for the monomer as measured by the device. Consequently, this method would be much better suited to studying the degradation of PBPAC, or any other polymer, under conditions that favor an action in exo, as can quite often be observed with enzymes. This is even more true when, instead of focusing on the level of polymer degradation, the emphasis is placed on the production of a specific species (i.e. monomer or degradation product) for its subsequent valorization. Figure : HPLC chromatograms obtained from PBPAC methanolysis of after 0, 2, 5, 15, 30 and 120 minutes of reaction.