A drawback of NIR fluorophores such as SWCNTs are their lower quantum yields. Interestingly, certain covalent modifications of the graphene lattice (sp quantum defects) create highly emissive states that increase the overall quantum yield . These quantum defects act as traps for mobile excitons from which red-shifted emission (E11*) takes place . Possible reactions to introduce defects include (a) ozone treatment , (b) reactions with aryldiazonium salts, which introduce sp 3 defects into the sp 2 lattice 28 and (c) [2π + 2π] photocycloaddition of enones , which all generate a redshifted photoluminescence around 1130 nm for (6,5)-SWCNTs. In addition to the reactions mentioned above, there are other ways to introduce covalently functional groups such as π-preserving triazine conjugation but without new emission features . Diazonium chemistry also offers to conjugate more complex entities such as biomolecules . It is useful to directly attach e.g. nanobodies or grow peptide chains on the SWCNT lattice, which expands the toolbox for many applications such as sensing with quantum defects . The concentration of diazonium salt affects the reaction and most likely the density of quantum defects. the ratio (E11 * /E11) between the quantum defect related emission (E11 * ) and the bandgap emission (E11) of SWCNTs is regarded as an approximate measure for defect density . Wang et al. estimated in SWCNTs of maximum E11 * photoluminescence about one defect per 20 nm length of (6,5)-SWCNTs . Zaumseil et al. found defect densities ranging from around 5 (low) to 30 (high) defects per µm of SWCNT . For (6,5)-SWCNTs, they recently measured sp 3 defects from low to medium densities at low temperatures (4.6 K), which equate to 4 µm -1 and 8 µm -1 , respectively, with an error of ±3 defects/µm . Nevertheless, a complete understanding of how defects affect photophysics is still missing. Even though there have been advances in synthesis of chirality-pure SWCNTs , commercially available SWCNTs typically contain different chiralities. Such mixtures of multiple SWCNT chiralities lead to overlapping and congested spectra. In contrast, purified SWCNTs show well-defined spectra and higher fluorescence emission . These advantages make chirality-enriched or pure SWCNTs highly attractive and are required to get well-defined SWCNT spectra e.g. for ratiometric or multiplexed sensing . In the past years, the efficacy at which SWCNTs can be dispersed, enriched, and separated has significantly improved . Purification methods include ion-exchange chromatography (IEX) , density-gradient ultracentrifugation (DGU) , selective extraction in organic solvents , gel chromatography and aqueous two-phase extraction (ATPE) . ATPE stands out among these methods, because it is relatively simple, provides a high reproducibility and up-scaling is straightforward. Zheng et al. reported an ATPE system which achieved complete sorting of DNA-SWCNTs 62 . Flavel and coworkers set up a simple pH sensitive ATPE process. In total 11 (n, m) species were isolated, not only including semiconducting but also metallic and non-armchair metallic species . The partition mechanism of SWCNTs chiralities in ATPE is still not completely understood but one plausible explanation are differences in the chemical potential for different (n, m) species in these two phases . Additionally, ATPE paired with surfactant exchange expands the potential for biological applications of SWCNTs . Here, we combine ATPE of SWCNTs with quantum defect chemistry to quantify the number of quantum defects in single SWCNTs. For this purpose, we apply diazonium salt chemistry and elucidate the impact of defects on the overall ATPE process. Furthermore, we follow the reaction of monochiral (6,5)-SWCNTs on the single molecule and single SWCNT level by fluorescence microscopy. This allows us to count the number of quantum defects and compare the bulk and single particle picture. To create SWCNTs with quantum defects (dSWCNTs), diazonium salt (4-nitrobenzoldiazonium tetrafluoroborate) was added to a sodium dodecyl benzenesulfonate (SDBS)dispersed SWCNT solution and exposed to green light (550 nm, Figure ). It is known that excessive amounts of sp 3 defects completely quench the NIR fluorescence of SWCNTs . Therefore, the reaction conditions were optimized first. In the absence of 4-nitrobenzoldiazonium tetrafluoroborate, there is only one E11 emission peak at 976 nm (figure ). With increasing concentration of diazonium salt, a new defect-related emission peak E11 * appeared at 1120 nm. Its intensity increased with diazonium salt concentration, while the intensity of the E11 peak decreased. With higher concentrations of the diazonium salt, the intensity of the E11 * emission decreased until the overall fluorescence vanished. Reaction mixtures with 10 µM, 20 µM and 30 µM diazonium salt (here after referred to as dlow-, dmedium-and dhigh-SWCNT) revealed the highest E11 and E11 * intensities and were used for further experiments. In the absorbance spectra there was no new peak observed at around 1120 nm (Figure ), which indicates a low density of quantum defects . The success of the defect reaction can be followed by an increase of the E11 * feature in both NIR 1D fluorescence (Figure ), Raman (Figure ) and 2D (Figure , b) excitation-emission fluorescence spectra. Additionally, the 2D spectra revealed that the prepared pristine-and dlow-SWCNT solutions consisted not only of (6,5)-SWCNTs but also of other SWCNT chiralities. A reliable and easy ATPE approach was used to enrich monochiral (6,5)-pristine-SWCNTs and (6,5)-dlow-SWCNTs and all steps were monitored by absorbance spectra of the respective top and bottom phases (Figure and). In the first step, HCl was added, which moved SWCNTs with larger diameter to the top phase 1 (T1), and mainly (6,5), (6,4) chiralities remained in the bottom phase 1 (B1). In the second step of ATPE, only (6,5) moved to the top phase 2 (T2) and all other chiralities remained in the bottom phase 2 (B2). In the last step, a separation of metallic and semiconducting SWCNTs was achieved and either (6,5)-pristine-SWCNTs or (6,5)-dlow-SWCNTs were collected in bottom phase 3 (B3). During ATPE, monochiral dlow-SWCNTs could be extracted in the same way as monochiral pristine-SWCNTs, as can be seen by the presence of chirality-specific absorbance peaks in the spectra of each phase in each step (Figure and S4). After ATPE, the absorbance and fluorescence spectra of SWCNTs showed mainly the E11 peak (around 986 nm) of pure (6,5)-SWCNTs (Figure , 2c and 2d). Similarly, well-defined spectra were obtained for dlow-SWCNTs after ATPE. In the absorbance spectrum only the expected features such as the E11 and E22 peaks were present (Figure ). Well-defined E11 and E11 * peaks were also observed in 1D (Figure ) and 2D (Figure ) excitationemission fluorescence spectra. These results show that the ATPE protocol separates monochiral (6,5)-SWCNTs with quantum defects from a mixture of different chiralities. Furthermore, the effect of diazonium salt concentration was explored. The E11 * /E11 ratio increased in NIR 1D and 2D excitation-emission fluorescence spectra (Figure , 2b, S5a, and S5b), which indicates increased densities of quantum defects before ATPE. During ATPE, the top and bottom phase absorbance spectra from all steps are the same (Figure S4, S6, and S7), suggesting that the same chiralities were separated regardless of defect density. After ATPE, dmediumand dhigh-SWCNT spectra indicated pure (6,5)-dSWCNTs, comparable to dlow-SWCNTs (Figure and). The partition coefficient of pristine and SWCNTs with quantum defects into different phases was therefore identical. It can be explained by similar chemical potentials, which is supported e.g. by similar Zeta potentials of SWCNTs with different defect densities (Figure ). All these findings pinpoint to a low number of defects per SWCNT. Moreover, the yield (total SWCNT mass) of performing first the defect reaction and then ATPE for dSWCNTs (3.3 %) was similar to the one for ATPE of pristine SWCNTs followed by the defect reaction (around 2.7 %) and similar to literature values . The different optical features of pristine and defect (6,5)-SWCNTs (after ATPE) allow us to observe defects on the single SWCNT level (Figure ). For this purpose, NIR fluorescence images were taken in the spectral range of the E11 emission (< 1020 nm) and of the E11* emission (>1050 nm). This spectral separation was only possible because of the high purity of the samples after ATPE without other chiralities of SWCNTs that could overlap with these emission features (figure ). Interestingly, we observed a large heterogeneity on the single SWCNT level. Some SWCNTs showed only E11 signals (figure , SWCNT 1), some only E11 * (f) Height difference from all pairs in (e) and their occurrence show that for the first two quantum defects the absolute changes in intensity are similar. This makes sense in the limit of small defect numbers when they are most likely independent exciton traps. signals (figure , SWCNT 2) and some both signals (figure , SWCNT 3). On the other hand, by comparing the fluorescence intensities of E11 and E11 * on a single SWCNT level, the different defect densities of dSWCNTs became qualitatively and also quantitatively visible. This way the E11 * /E11 ratio of thousands of particles could be measured (figure ) and as expected, the mean ratio shifted to higher values with the increased defect densities (figure ). The ratio of E11 * /E11 was interpreted to change linearly with the number of defects and the probability of defect numbers on one SWCNT was calculated with a Poisson probability mass function: