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Synthesis and spectral measurements of sulphonated graphene: some anomalous observations† Susmita Maiti, Somashree Kundu, Debasmita Ghosh, Somrita Mondal, Chandra Nath Roy and Abhijit Saha* The present report demonstrates how a sulphonation process, a key route for synthesizing water soluble graphene, can influence the optical behavior of precursor graphene oxide, intermediate reaction products and sulphonated graphene. We observed that there is constant emission maximum at 500 nm for graphene oxide in the excitation range of 320–450 nm. During sulphonation, sulphonated reduced graphene oxide (rGO-SO3H) is initially formed which has an emission at 358 nm. However, the reduction of oxygen containing groups in rGO-SO3H with hydrazine hydrate leading to the formation of SG caused a shift in the emission to 430 nm, which has been attributed to the extended delocalization of p-electrons involving the phenyl sulphonate group.
Received 28th September 2015, Accepted 29th January 2016
In the present investigation, we have identified many existing anomalies in the important spectral features of
DOI: 10.1039/c5cp05799j
these materials, such as violation of Kasha’s rule on fluorescence and pH dependence emission. Furthermore, it has also been shown that proper care is necessary to be taken in monitoring the fluorescence of sulphonated
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graphene in view of possible interference from the components produced during sulphonation.
1. Introduction Graphene is a perfect single-layered sheet in which every carbon atom is sp2 hybridized and p-conjugated with adjacent carbons. It is a zero band gap semiconductor and is not expected to be photoactive or photoluminescent. However, since its discovery in 2004,1 the 2D nanostructure and chemical functionality of graphene materials have made it highly attractive in optoelectronics,2 catalysis, drug delivery,3 and cell imaging4,5 as well as chemo/biosensing.6 Graphene oxide (GO) and the corresponding chemically reduced GO (rGO) are actually chemically functionalized graphene that retain many properties of the pure graphene. In recent years, interest in carbon-based fluorescent materials has increased. Compared to conventional materials, carbon based ones often show greater stability, biocompatibility and lower cytotoxicity properties, which are of great advantage in biological applications.7 The origin of photoluminescence (PL) in these carbon-based nanomaterials is tentatively proposed to be from isolated polyaromatic structures or passivated surface defects.8 However, the preparation of these carbon nanomaterials usually shows a very low yield and is carried out under extreme conditions,
UGC-DAE Consortium for Scientific Research, Kolkata Centre, Kolkata 700098, India. E-mail: [email protected]; Fax: +91-33-23357008; Tel: +91-33-23351866 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5cp05799j
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e.g. laser ablation, high temperature and high pressure.8 A graphene oxide (GO) nanosheet, a two-dimensional oxidized derivative of graphene, also contains isolated polyaromatic clusters and can be easily exfoliated from graphite with a high yield under simple oxidizing conditions. The photoluminescence properties of GO may originate from the recombination of electron–hole pairs generated within the localized graphitic sp2 states arising from the various defect structures produced from the oxidation of graphite to GO. However, there are different propositions on the emission of GO.9–13 Recently, an extremely weak broad PL of GO was reported, which was believed to originate from the carbon sp2 domains/clusters.14 Even though the PL intensity was slightly improved after moderate reduction using hydrazine, the quantum yield (QY) was too low to be measured accurately.14 Moreover, the weak PL was quenched upon further reduction with hydrazine due to the formation of nonfluorescent graphene that is known as a zero-bandgap semiconductor. Furthermore, the pH-dependent PL properties of GO have recently been revealed,15–17 showing a new pathway to develop different sensors.18–20 However, in recent years, graphene has shown tremendous promise for the applications in the biological arena. Since, biological systems predominantly work in an aqueous environment having cellular water content of approximately 80%, it is imperative to make graphene or its derivatives water soluble. Hence, there is growing demand for the synthesis of water soluble graphene. Si and Samulski21 first made graphene
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soluble through sulphonation by coupling sulphanilic acid to graphene molecules. In addition, the oxide of graphene molecules is also soluble in water and retains the basic structure of the graphene sheet although there are some differences in the nature of the functional groups present. This has spurred interest in the investigation of the fluorescence behaviour of these materials. Thus, we have synthesized sulphonated graphene (SG) from graphene oxide by following the method of Si and Samulski21 with some modifications and endeavoured systematically to look into the fluorescence patterns of GO, SG and also the intermediate products formed in the process of sulphonation, like rGO and rGO-SO3H. Recently, it has been reported that graphene oxide shows interesting emission behaviour through the strong excitation wavelength dependent fluorescence originated from the ‘‘giant red-edge effect’’ in polar medium, breaking Kasha’s rule.28 This has prompted us to monitor whether all the graphene based materials as mentioned earlier display excitation dependent fluorescence. We have repeated the absorption and fluorescence measurements several times on all the four materials even at different temperatures. However, our observed results suggest that all the graphene compounds both in polar and apolar media obeys Kasha’s rule. Thus, the report on the excitation wavelength dependent fluorescence of GO breaking Kasha’s rule appears to be debatable. We have also furnished evidence that appropriate care should be taken in following the fluorescence of SG, since sulphanilic acid used for sulphonation can interfere in the emission of SG. In addition, it has been shown that the by-product which may be formed upon hydrazine reduction of a diazotized salt of sulphanilic acid has overlapping emission with the fluorescence of SG. We have also examined the pH dependence on the fluorescence of SG as reported by Kundu et al.22 It is observed that they have wrongly used commercial buffer capsules to maintain the pH of SG and these commercial capsules can show fluorescence depending on the constituent of the buffer capsules of respective pH. Thus, the pH dependent fluorescence of SG as shown by Kundu et al.22 is apparently inappropriate. The present investigation therefore provides a rational estimate of fluorescence behaviour of watersoluble graphene materials.
2. Experimental 2.1.
Materials
Graphite flakes (natural, 325 mesh, 99.8%) were purchased from Alfa Aesar. Sodium nitrate (NaNO3), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), sulphuric acid (H2SO4), sulphanilic acid, sodium nitrate, 35% hydrochloric acid (G.R. grade), and hydrazine hydrate (99%, synthetic grade) were purchased from Merck, India. All chemicals used were of analytical grade or of high purity available. Milli-Q water (Millipore) was used as a solvent. 2.1.1 Synthesis of graphene oxide and sulphonated graphene. At first graphene oxide (GO) was synthesized by the chemical exfoliation of graphite which was accomplished by the oxidative treatment of natural graphite using Hummers’ method23, followed by facile exfoliation via sonication.21 In brief, graphite flakes (0.5 g),
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sodium nitrate (0.25 g), and sulphuric acid (12 mL) were mixed in an ice bath with vigorous stirring. Then 1.5 g of potassium permanganate was slowly added to the mixture and temperature was maintained at less than 20 1C. Subsequently, the ice bath was removed and the mixture was kept at a temperature of 35 5 1C for half an hour. The mixture appeared as a thick paste. After that about 25 mL of water was added to it and it was left for 15 minutes. The temperature rose to 90 5 1C during the addition of water. The solution was further diluted with 100 mL of water. 30% H2O2 solution was subsequently added to it and its colour became bright yellow. The solution was then filtered under warm conditions. The residue of graphite oxide was re-dispersed in 150 mL of water and centrifuged several times and dried in a vacuum. The graphite oxide so obtained was dispersed in water and sonicated with about 25 kHz frequency and a power of 650 W for 1 hour resulting in the formation of a clear brown dispersion of graphene oxide. However, if the oxygen functionality is removed to yield graphene, the graphene sheets lose their water dispersibility, aggregate, and eventually precipitate. In order to overcome this, we introduce p-phenyl-SO3H groups into the graphene oxide before it is fully reduced and the resulting graphene remains soluble in water and does not aggregate. There are three steps in the synthesis of SG from GO: (1) pre-reduction of GO with sodium borohydride, (2) sulphonation with the aryl diazonium salt of sulphanilic acid, (3) postreduction with hydrazine. At first, 800 mg of sodium borohydride solution in 15 mL of water was added into 75 mL of the GO dispersion in water and its pH was adjusted at 9–10 by the addition of 5 wt% sodium carbonate solution. The mixture was then heated and was maintained at 100 1C for 2 h under constant stirring. Then it was centrifuged and washed several times with Mili-Q water to get rGO. To prepare rGO-SO3H, rGO was redispersed in 75 mL of water by diazonium coupling reaction. For this purpose, 52 mg of sulphanilic acid was dissolved in water and 26 mg of sodium nitrite was dissolved in 10 mL of water. Then 0.5 mL of 1 M HCl under ice cooled conditions was added to the sodium nitrite solution. After that this resulting solution was added to the sulphanilic acid solution under ice cooled conditions. Then this diazotized salt was added to the reduced graphene oxide solution and was kept for 4 h under constant stirring. It was centrifuged and washed repeatedly with water until it became neutral. The product was then redispersed in 50 mL of water and was reduced with 20 mL of hydrazine hydrate solution, 20 mL of water and 35 mL of ammonia solution (25% in water, Merck, India) under reflux conditions for 48 h at 100 1C. Finally, it was washed with water thoroughly and dried in a vacuum to get SG. Fourier transform infra-red (FTIR) spectra, Raman spectra, X-ray diffraction (XRD) pattern, energy dispersive spectrum (EDS) of SG and transmission electron microscopy (TEM) images of the synthesized graphene oxide and SG are presented in the ESI.† 2.2.
Methods
UV-Visible absorption spectra were recorded using a Perkin Elmer (Lambda 950) UV-Vis-NIR spectrophotometer. Baseline corrections were done using Milli-Q water. Quartz cells of 1 cm optical path were used as sample holders.
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Photoluminescence (PL) measurements were performed at room temperature using a Perkin Elmer (LS 55) luminescence spectrometer in water and buffer medium. Fluorescence lifetimes were determined from time-resolved intensity decays at various wavelengths by using a time-correlated-single photoncounting (TCSPC) spectrophotometer (Edinburgh) with a lamp of FWHM = 1.2 ns, and a repetition rate of 25 kHz. The details of the set-up were described elsewhere.24 For GO, excitation was done at 377 nm and the emission was recorded at 520, 580, 620 and 680 nm. The quality of the fits is judged by the reduced w2 criterion. Mean (average) fluorescence lifetimes (tav) for the bi-exponential fittings are calculated from the decay times (t1 and t2) and the relative amplitudes (a1 and a2) using the relation (1) hti ¼
n X Ai ti2 i¼1
Ai ti
(1)
FTIR spectroscopic measurements of GO and SG were recorded using a FTIR spectrometer (Perkin Elmer, Spectra GX). Samples for FTIR measurements were prepared in the form of pellets by mixing 200 mg of IR spectroscopic grade potassium bromide with 2 mg of dried sample (i.e., GO and SG). The spectra were recorded in a transmission mode over 20 scans with a resolution of 4 cm1. The Raman spectra of graphene oxide (GO) and SG were recorded using a Lab Ram HR 800 (Horiba Jobin Yvon) spectrometer. The instrument acquired data over a range of 200 cm1 to 3500 cm1 with an exposure time of 5 s. The laser power was 17 mW and the operating wavelength of the He–Ne laser was 632.8 nm. Spectral detection was obtained through the use of a Peltier cooled charge-coupled device (CCD) detector. The samples for Raman spectral measurements were prepared in the form of films on a glass and were excited using the laser. TEM images were taken on electron microscopes (JEOL-2010 and FEI Tecnai S-twin) operated at an acceleration voltage of 200 kV. A drop of aqueous solution of GO and SG was placed on a carbon-coated copper grid of 400 mesh and allowed to soak. Then, the grids were kept overnight in a vacuum desiccator to dry properly. X-ray diffraction measurements were performed using a Bruker D8 Advance X-ray diffractometer operated at a current of 40 milli Amp and 40 kV voltage. The scan rate was 21 minute1 in the 2y range of 10–801 during data accumulation. The Cu Ka (l = 0.1546 nm) was used as the radiation source for the experimental measurements. Sonication was done by using Ultrasonic Homogenizer (Model: U650) Takashi, Japan.
Fig. 1
UV-Visible absorption spectra of GO, rGO, rGO-SO3H and SG.
assumed that disappearance of the 306 nm band could be due to the removal of some of the oxygenated functional groups, such as carboxyl, epoxy and carbonyl groups, which in turn, facilitates the p-electron delocalization in rGO leading to the observed red shift of absorption maximum. Again, we observe that sulphonation has resulted in red shift of absorption of rGO at 255 nm to 272 nm in rGO-SO3H and 269 nm in SG. This red shift can be envisaged as the possible extended p-electron delocalization in the graphene system due to the incorporation of the sulphonated phenyl ring and additional reduction by hydrazine hydrate. The photoluminescence spectra of GO in water at different excitation wavelengths are presented in Fig. 2. A typical fluorescence decay profile is shown in the Fig. 2 inset. The average fluorescence lifetime of GO was around 0.5 ns. The emission peak appears around 505 nm under various excitation wavelengths (320–450 nm). Thus, it is observed that the fluorescence emission from graphene oxide does not depend on excitation energy at room temperature. This is in contradiction to the shift of emission maxima with varying excitation photons in the similar range to that reported by Cushing et al.27 Their observation, in fact, breaks Kasha’s rule,
3. Results and discussion Fig. 1 illustrates the UV-vis absorption spectra of GO, r-GO, r-GO-SO3H, SG in the wavelength range of 200–400 nm. GO shows an absorption peak at 235 nm and a shoulder at 306 nm, which have been ascribed earlier to the p–p* transition of sp2 hybrid regions of C–C and CQC bonds and the n–p* transition of the CQO bond, respectively.25,26 It is observed that maximum absorption peak of GO at 235 nm is red shifted to 255 nm and the shoulder disappeared on the reduction of GO to rGO. It may be
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Fig. 2 PL spectra of GO in water with different excitations and inset: a fluorescence decay profile with lex at 377 and lem at 520 nm.
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which states that fluorescence in a molecular system always occurs from the lowest singlet state implying excitation independent emission.28 In semiconducting materials, all excited electrons, independent of initial energy, relax to the band edge before fluorescence proceeds. However, Shang et al. demonstrated, like ours, constant emission maximum in the excitation range of 350–450 nm and red shifts in emission only at higher excitation wavelengths greater than 450 nm (Fig. S6, ESI†) due to the presence of multiple chromophores/fluorophores involved with aromatic and oxidation groups in the graphene oxide system.17 Furthermore, Kozawa et al.29 showed that there could be two emissions with excitations at 220 and 330 nm, which were attributed to the presence of different functional moieties such as hydroxybenzoic acid and dicarboxybenzoic acid. Therefore, violation of Kasha’s rule in the graphene oxide system as claimed by Cushing et al. appears to be controversial and debatable. The emission spectra of rGO and rGO-SO3H are shown in Fig. 3. Interestingly, the emission peak appears at 358 nm for both rGO and rGO-SO3H upon excitation at 240–270 nm. This shows that the sulphonation of reduced graphene oxide does not alter the emission profile. The observed results suggest that the origin of fluorescence in rGO remains unaltered with the introduction of the phenyl sulphonic acid group. However, sulphonated graphene produced from rGO-SO3H through reduction with hydrazine hydrate shows the emission maximum at 430 nm with an excitation of 310–340 nm. Therefore, the observed results clearly demonstrate that the reduction of oxygenated groups generates different fluorescence centers. It may be assumed that the reduction of oxygen containing groups may facilitate the extended delocalization of p-electrons involving the phenyl sulphonate group, which leads to the red-shift of fluorescence emission in SG to 430 nm from 358 nm of rGO-SO3H. We have followed the pH dependence of the fluorescence of GO and SG. The pH of the solution was appropriately adjusted with 0.1 M HCl or 0.1 M NaOH. We observed that the variation of pH in the range 4–9 had no influence on the spectral nature. However, Kundu et al.22 showed the change in the fluorescence spectra with pH. In our opinion, the main drawback in their experimental design was that pH of the solution was
Fig. 3
PL spectra of r-GO and rGO-SO3H (inset) with different excitations.
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Fig. 4 PL spectra of SG with different excitations in water (pH 6.5) and inset: PL spectra of SG dissolved in commercial buffers of pH 4 and pH 9.2.
maintained with commercially available buffer capsules of respective pH. We have checked the fluorescence of commercial buffer capsules and found that these capsules display fluorescence at the similar position to that reported in the respective pH. The emission spectrum of SG in water at pH 6.5 is shown in Fig. 4. The PL spectra of GO and SG recorded at pH 4 and 9.2 in the wavelength range of 350–550 nm under excitation at 320 nm (maintained with buffer capsules) are also presented (inset). For SG in pH 4 the emission maximum occurs at around 435 nm, which is also the emission peak position of buffer. We observe that appropriate care should be taken when emission of SG is followed. Excitation with 250 nm can be a problem as a strong emission appears at 350 nm, since we identify that this peak is primarily due to phenyl sulphonic acid used for sulphonation.30 The emission spectra of blank sulphanilic acid are presented in the inset of Fig. 5. Thus, to overcome this interference, in obtaining pure emission of SG, excitation wavelengths 310–340 nm should be used, as there is no absorption of pure sulphanilic acid in this
Fig. 5 PL spectra of reaction mixture excited at different wavelengths in the absence of graphene materials. Inset: Emission of sulphanilic acid with different excitations.
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Scheme 1
Paper
Reaction products of hydrazine hydrate with sulphanilic acid.
range. Therefore, with this excitation, the emission peak appearing around 430 nm (Fig. 4) is exclusively that of the emission spectrum of sulphonated graphene.16 Furthermore, we followed the possible reaction products of hydrazine hydrate with sulphanilic acid (Scheme 1) in the absence of GO under the similar experimental conditions. Surprisingly, we find that there are two emission peaks at 350 nm and 450 nm in the reaction mixture (Fig. 5). It is apparent that the peaks at 350 and 450 nm are due to unreacted phenyl sulphonic acid and sulphonated phenyl hydrazine, produced respectively. However, this product being highly water soluble is removed by proper washing of the synthesized SG and thus, ensures no interference in spectral measurements of sulphonated graphene.
4. Conclusions In conclusion, the synthesis of water soluble graphene and accurate determination of its fluorescence characteristics are of utmost importance for the use of graphene based materials in various bio-medical applications, such as sensors, imaging devices, drugcarriers, etc. Water soluble sulphonated graphene and intermediate compounds/materials formed upon sulfonation of graphene oxide were analyzed through spectroscopic techniques. The present report reveals that violation of Kasha’s rule and pH dependence on spectral features of graphene materials as claimed recently are controversial and debatable, which may have serious ramifications in the growing applications of the graphene-based materials. We have also pointed out that proper care should be taken in monitoring the fluorescence of sulphonated graphene in view of possible interference from the components produced during sulphonation.
Acknowledgements The authors wish to thank IIT Kharagpur for providing the TEM facility. One of the authors (S. Maiti) is thankful to DST, Govt. of India, for the award of the INSPIRE fellowship. Two of the authors (S. Kundu and S. Mondal) are thankful to the UGC, Govt. of India for a NET-Junior Research Fellowship. Another author (D. Ghosh) is thankful to CSIR, Govt. of India for a Senior Research Fellowship.
References 1 K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. Dubonos, I. V. Grigorieva and A. A. Firsov, Science, 2004, 306, 666–669.
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2 F. Bonaccorso, Z. Sun, T. Hasan and A. C. Ferrari, Nat. Photonics, 2010, 4, 611–622. 3 G. M. Scheuermann, L. Rumi, P. Steurer, W. Bannwarth and R. Mulhaupt, J. Am. Chem. Soc., 2009, 131, 8262–8270. 4 X. Sun, Z. Liu, K. Welsher, J. Robinson, A. Goodwin, S. Zaric and H. Dai, Nano Res., 2008, 1, 203–212. 5 Z. Liu, J. T. Robinson, X. Sun and H. Dai, J. Am. Chem. Soc., 2008, 130, 10876–10877. 6 C. H. Lu, H. H. Yang, C. L. Zhu, X. Chen and G. N. Chen, Angew. Chem., Int. Ed., 2009, 48, 4785–4787. 7 S. Zhu, S. Tang, J. Zhang and B. Yang, Chem. Commun., 2012, 48, 4527–4539. 8 J. E. Riggs, Z. X. Guo, D. L. Carroll and Y. P. Sun, J. Am. Chem. Soc., 2000, 122, 5879. 9 Q. Mei, K. Zhang, G. Guan, B. Liu, S. Wang and Z. Zhang, Chem. Commun., 2010, 46, 7319–7321. 10 K. S. Subrahmanyam, P. Kumar, A. Nag and C. N. R. Rao, Solid State Commun., 2010, 150, 1774–1777. 11 G. Eda, Y. Lin, C. Mattevi, H. Yamaguchi, H. Chen, I. Chen, C. Chen and M. Chhowalla, Adv. Mater., 2010, 22, 505–509. 12 K. P. Loh, Q. Bao, G. Eda and M. Chhowalla, Nat. Chem., 2010, 2, 1015–1024. 13 W. Zhao, C. Song and P. E. Pehrsson, J. Am. Chem. Soc., 2002, 124, 12418–12419. 14 Z. T. Luo, P. M. Vora, E. J. Mele, A. T. Charlie Johnson and J. M. Kikkawa, Appl. Phys. Lett., 2009, 94, 111909. 15 J. L. Chen and X. P. Yan, Chem. Commun., 2011, 47, 3135–3137. 16 C. Galande, A. D. Mohite, A. V. Naumov, W. Gao, L. Ci, A. Ajayan, H. Gao, A. Srivastava, R. B. Weisman and P. M. Ajayan, Sci. Rep., 2011, 1(85), 1–5. 17 J. Shang, L. Ma, J. Li, W. Ai, T. Yu and G. G. Gurzadyan, Sci. Rep., 2012, 2(792), 1–8. 18 J. L. Chen, X. P. Yan, K. Meng and S. F. Wang, Anal. Chem., 2011, 83, 8787–8793. 19 A. Kundu, R. K. Layek and A. K. Nandi, J. Mater. Chem., 2012, 22, 8139–8144. 20 A. Kundu, R. K. Layek, A. Kuila and A. K. Nandi, ACS Appl. Mater. Interfaces, 2012, 4, 5576–5582. 21 Y. Si and E. T. Samulski, Nano Lett., 2008, 8(6), 1679–1682. 22 A. Kundu, S. Nandi, R. K. Layek and A. K. Nandi, ACS Appl. Mater. Interfaces, 2013, 5, 7392–7399. 23 W. S. Hummers and R. E. Offeman, J. Am. Chem. Soc., 1958, 80, 1339. 24 S. Aich, C. Raha and S. Basu, J. Chem. Soc., Faraday Trans., 1997, 93, 2991–2996. 25 T. V. Cuong, V. H. Pham, Q. T. Tran, S. H. Hahn, J. S. Chung, E. W. Shin and E. J. Kim, Mater. Lett., 2010, 64, 399–401. 26 Z. Luo, Y. Lu, L. A. Somers and A. T. C. Johnson, J. Am. Chem. Soc., 2009, 131, 898–899. 27 S. K. Cushing, M. Li, F. Huang and N. Wu, ACS Nano, 2014, 8, 1002–1013. 28 M. Kasha, Discuss. Faraday Soc., 1950, 9, 14–19. 29 D. Kozawa, Y. Miyauchi, S. Mouri and K. Matsuda, J. Phys. Chem. Lett., 2013, 4, 2035–2040. 30 K. A. Connors, A text book of pharmaceutical analysis, John Wiley & Sons, India, 2007.
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Synthesis and spectral measurements of sulphonated graphene: some anomalous observations† Susmita Maiti, Somashree Kundu, Debasmita Ghosh, Somrita Mondal, Chandra Nath Roy and Abhijit Saha* The present report demonstrates how a sulphonation process, a key route for synthesizing water soluble graphene, can influence the optical behavior of precursor graphene oxide, intermediate reaction products and sulphonated graphene. We observed that there is constant emission maximum at 500 nm for graphene oxide in the excitation range of 320–450 nm. During sulphonation, sulphonated reduced graphene oxide (rGO-SO3H) is initially formed which has an emission at 358 nm. However, the reduction of oxygen containing groups in rGO-SO3H with hydrazine hydrate leading to the formation of SG caused a shift in the emission to 430 nm, which has been attributed to the extended delocalization of p-electrons involving the phenyl sulphonate group.
Received 28th September 2015, Accepted 29th January 2016
In the present investigation, we have identified many existing anomalies in the important spectral features of
DOI: 10.1039/c5cp05799j
these materials, such as violation of Kasha’s rule on fluorescence and pH dependence emission. Furthermore, it has also been shown that proper care is necessary to be taken in monitoring the fluorescence of sulphonated
www.rsc.org/pccp
graphene in view of possible interference from the components produced during sulphonation.
1. Introduction Graphene is a perfect single-layered sheet in which every carbon atom is sp2 hybridized and p-conjugated with adjacent carbons. It is a zero band gap semiconductor and is not expected to be photoactive or photoluminescent. However, since its discovery in 2004,1 the 2D nanostructure and chemical functionality of graphene materials have made it highly attractive in optoelectronics,2 catalysis, drug delivery,3 and cell imaging4,5 as well as chemo/biosensing.6 Graphene oxide (GO) and the corresponding chemically reduced GO (rGO) are actually chemically functionalized graphene that retain many properties of the pure graphene. In recent years, interest in carbon-based fluorescent materials has increased. Compared to conventional materials, carbon based ones often show greater stability, biocompatibility and lower cytotoxicity properties, which are of great advantage in biological applications.7 The origin of photoluminescence (PL) in these carbon-based nanomaterials is tentatively proposed to be from isolated polyaromatic structures or passivated surface defects.8 However, the preparation of these carbon nanomaterials usually shows a very low yield and is carried out under extreme conditions,
UGC-DAE Consortium for Scientific Research, Kolkata Centre, Kolkata 700098, India. E-mail: [email protected]; Fax: +91-33-23357008; Tel: +91-33-23351866 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5cp05799j
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e.g. laser ablation, high temperature and high pressure.8 A graphene oxide (GO) nanosheet, a two-dimensional oxidized derivative of graphene, also contains isolated polyaromatic clusters and can be easily exfoliated from graphite with a high yield under simple oxidizing conditions. The photoluminescence properties of GO may originate from the recombination of electron–hole pairs generated within the localized graphitic sp2 states arising from the various defect structures produced from the oxidation of graphite to GO. However, there are different propositions on the emission of GO.9–13 Recently, an extremely weak broad PL of GO was reported, which was believed to originate from the carbon sp2 domains/clusters.14 Even though the PL intensity was slightly improved after moderate reduction using hydrazine, the quantum yield (QY) was too low to be measured accurately.14 Moreover, the weak PL was quenched upon further reduction with hydrazine due to the formation of nonfluorescent graphene that is known as a zero-bandgap semiconductor. Furthermore, the pH-dependent PL properties of GO have recently been revealed,15–17 showing a new pathway to develop different sensors.18–20 However, in recent years, graphene has shown tremendous promise for the applications in the biological arena. Since, biological systems predominantly work in an aqueous environment having cellular water content of approximately 80%, it is imperative to make graphene or its derivatives water soluble. Hence, there is growing demand for the synthesis of water soluble graphene. Si and Samulski21 first made graphene
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soluble through sulphonation by coupling sulphanilic acid to graphene molecules. In addition, the oxide of graphene molecules is also soluble in water and retains the basic structure of the graphene sheet although there are some differences in the nature of the functional groups present. This has spurred interest in the investigation of the fluorescence behaviour of these materials. Thus, we have synthesized sulphonated graphene (SG) from graphene oxide by following the method of Si and Samulski21 with some modifications and endeavoured systematically to look into the fluorescence patterns of GO, SG and also the intermediate products formed in the process of sulphonation, like rGO and rGO-SO3H. Recently, it has been reported that graphene oxide shows interesting emission behaviour through the strong excitation wavelength dependent fluorescence originated from the ‘‘giant red-edge effect’’ in polar medium, breaking Kasha’s rule.28 This has prompted us to monitor whether all the graphene based materials as mentioned earlier display excitation dependent fluorescence. We have repeated the absorption and fluorescence measurements several times on all the four materials even at different temperatures. However, our observed results suggest that all the graphene compounds both in polar and apolar media obeys Kasha’s rule. Thus, the report on the excitation wavelength dependent fluorescence of GO breaking Kasha’s rule appears to be debatable. We have also furnished evidence that appropriate care should be taken in following the fluorescence of SG, since sulphanilic acid used for sulphonation can interfere in the emission of SG. In addition, it has been shown that the by-product which may be formed upon hydrazine reduction of a diazotized salt of sulphanilic acid has overlapping emission with the fluorescence of SG. We have also examined the pH dependence on the fluorescence of SG as reported by Kundu et al.22 It is observed that they have wrongly used commercial buffer capsules to maintain the pH of SG and these commercial capsules can show fluorescence depending on the constituent of the buffer capsules of respective pH. Thus, the pH dependent fluorescence of SG as shown by Kundu et al.22 is apparently inappropriate. The present investigation therefore provides a rational estimate of fluorescence behaviour of watersoluble graphene materials.
2. Experimental 2.1.
Materials
Graphite flakes (natural, 325 mesh, 99.8%) were purchased from Alfa Aesar. Sodium nitrate (NaNO3), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), sulphuric acid (H2SO4), sulphanilic acid, sodium nitrate, 35% hydrochloric acid (G.R. grade), and hydrazine hydrate (99%, synthetic grade) were purchased from Merck, India. All chemicals used were of analytical grade or of high purity available. Milli-Q water (Millipore) was used as a solvent. 2.1.1 Synthesis of graphene oxide and sulphonated graphene. At first graphene oxide (GO) was synthesized by the chemical exfoliation of graphite which was accomplished by the oxidative treatment of natural graphite using Hummers’ method23, followed by facile exfoliation via sonication.21 In brief, graphite flakes (0.5 g),
Phys. Chem. Chem. Phys.
sodium nitrate (0.25 g), and sulphuric acid (12 mL) were mixed in an ice bath with vigorous stirring. Then 1.5 g of potassium permanganate was slowly added to the mixture and temperature was maintained at less than 20 1C. Subsequently, the ice bath was removed and the mixture was kept at a temperature of 35 5 1C for half an hour. The mixture appeared as a thick paste. After that about 25 mL of water was added to it and it was left for 15 minutes. The temperature rose to 90 5 1C during the addition of water. The solution was further diluted with 100 mL of water. 30% H2O2 solution was subsequently added to it and its colour became bright yellow. The solution was then filtered under warm conditions. The residue of graphite oxide was re-dispersed in 150 mL of water and centrifuged several times and dried in a vacuum. The graphite oxide so obtained was dispersed in water and sonicated with about 25 kHz frequency and a power of 650 W for 1 hour resulting in the formation of a clear brown dispersion of graphene oxide. However, if the oxygen functionality is removed to yield graphene, the graphene sheets lose their water dispersibility, aggregate, and eventually precipitate. In order to overcome this, we introduce p-phenyl-SO3H groups into the graphene oxide before it is fully reduced and the resulting graphene remains soluble in water and does not aggregate. There are three steps in the synthesis of SG from GO: (1) pre-reduction of GO with sodium borohydride, (2) sulphonation with the aryl diazonium salt of sulphanilic acid, (3) postreduction with hydrazine. At first, 800 mg of sodium borohydride solution in 15 mL of water was added into 75 mL of the GO dispersion in water and its pH was adjusted at 9–10 by the addition of 5 wt% sodium carbonate solution. The mixture was then heated and was maintained at 100 1C for 2 h under constant stirring. Then it was centrifuged and washed several times with Mili-Q water to get rGO. To prepare rGO-SO3H, rGO was redispersed in 75 mL of water by diazonium coupling reaction. For this purpose, 52 mg of sulphanilic acid was dissolved in water and 26 mg of sodium nitrite was dissolved in 10 mL of water. Then 0.5 mL of 1 M HCl under ice cooled conditions was added to the sodium nitrite solution. After that this resulting solution was added to the sulphanilic acid solution under ice cooled conditions. Then this diazotized salt was added to the reduced graphene oxide solution and was kept for 4 h under constant stirring. It was centrifuged and washed repeatedly with water until it became neutral. The product was then redispersed in 50 mL of water and was reduced with 20 mL of hydrazine hydrate solution, 20 mL of water and 35 mL of ammonia solution (25% in water, Merck, India) under reflux conditions for 48 h at 100 1C. Finally, it was washed with water thoroughly and dried in a vacuum to get SG. Fourier transform infra-red (FTIR) spectra, Raman spectra, X-ray diffraction (XRD) pattern, energy dispersive spectrum (EDS) of SG and transmission electron microscopy (TEM) images of the synthesized graphene oxide and SG are presented in the ESI.† 2.2.
Methods
UV-Visible absorption spectra were recorded using a Perkin Elmer (Lambda 950) UV-Vis-NIR spectrophotometer. Baseline corrections were done using Milli-Q water. Quartz cells of 1 cm optical path were used as sample holders.
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Photoluminescence (PL) measurements were performed at room temperature using a Perkin Elmer (LS 55) luminescence spectrometer in water and buffer medium. Fluorescence lifetimes were determined from time-resolved intensity decays at various wavelengths by using a time-correlated-single photoncounting (TCSPC) spectrophotometer (Edinburgh) with a lamp of FWHM = 1.2 ns, and a repetition rate of 25 kHz. The details of the set-up were described elsewhere.24 For GO, excitation was done at 377 nm and the emission was recorded at 520, 580, 620 and 680 nm. The quality of the fits is judged by the reduced w2 criterion. Mean (average) fluorescence lifetimes (tav) for the bi-exponential fittings are calculated from the decay times (t1 and t2) and the relative amplitudes (a1 and a2) using the relation (1) hti ¼
n X Ai ti2 i¼1
Ai ti
(1)
FTIR spectroscopic measurements of GO and SG were recorded using a FTIR spectrometer (Perkin Elmer, Spectra GX). Samples for FTIR measurements were prepared in the form of pellets by mixing 200 mg of IR spectroscopic grade potassium bromide with 2 mg of dried sample (i.e., GO and SG). The spectra were recorded in a transmission mode over 20 scans with a resolution of 4 cm1. The Raman spectra of graphene oxide (GO) and SG were recorded using a Lab Ram HR 800 (Horiba Jobin Yvon) spectrometer. The instrument acquired data over a range of 200 cm1 to 3500 cm1 with an exposure time of 5 s. The laser power was 17 mW and the operating wavelength of the He–Ne laser was 632.8 nm. Spectral detection was obtained through the use of a Peltier cooled charge-coupled device (CCD) detector. The samples for Raman spectral measurements were prepared in the form of films on a glass and were excited using the laser. TEM images were taken on electron microscopes (JEOL-2010 and FEI Tecnai S-twin) operated at an acceleration voltage of 200 kV. A drop of aqueous solution of GO and SG was placed on a carbon-coated copper grid of 400 mesh and allowed to soak. Then, the grids were kept overnight in a vacuum desiccator to dry properly. X-ray diffraction measurements were performed using a Bruker D8 Advance X-ray diffractometer operated at a current of 40 milli Amp and 40 kV voltage. The scan rate was 21 minute1 in the 2y range of 10–801 during data accumulation. The Cu Ka (l = 0.1546 nm) was used as the radiation source for the experimental measurements. Sonication was done by using Ultrasonic Homogenizer (Model: U650) Takashi, Japan.
Fig. 1
UV-Visible absorption spectra of GO, rGO, rGO-SO3H and SG.
assumed that disappearance of the 306 nm band could be due to the removal of some of the oxygenated functional groups, such as carboxyl, epoxy and carbonyl groups, which in turn, facilitates the p-electron delocalization in rGO leading to the observed red shift of absorption maximum. Again, we observe that sulphonation has resulted in red shift of absorption of rGO at 255 nm to 272 nm in rGO-SO3H and 269 nm in SG. This red shift can be envisaged as the possible extended p-electron delocalization in the graphene system due to the incorporation of the sulphonated phenyl ring and additional reduction by hydrazine hydrate. The photoluminescence spectra of GO in water at different excitation wavelengths are presented in Fig. 2. A typical fluorescence decay profile is shown in the Fig. 2 inset. The average fluorescence lifetime of GO was around 0.5 ns. The emission peak appears around 505 nm under various excitation wavelengths (320–450 nm). Thus, it is observed that the fluorescence emission from graphene oxide does not depend on excitation energy at room temperature. This is in contradiction to the shift of emission maxima with varying excitation photons in the similar range to that reported by Cushing et al.27 Their observation, in fact, breaks Kasha’s rule,
3. Results and discussion Fig. 1 illustrates the UV-vis absorption spectra of GO, r-GO, r-GO-SO3H, SG in the wavelength range of 200–400 nm. GO shows an absorption peak at 235 nm and a shoulder at 306 nm, which have been ascribed earlier to the p–p* transition of sp2 hybrid regions of C–C and CQC bonds and the n–p* transition of the CQO bond, respectively.25,26 It is observed that maximum absorption peak of GO at 235 nm is red shifted to 255 nm and the shoulder disappeared on the reduction of GO to rGO. It may be
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Fig. 2 PL spectra of GO in water with different excitations and inset: a fluorescence decay profile with lex at 377 and lem at 520 nm.
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which states that fluorescence in a molecular system always occurs from the lowest singlet state implying excitation independent emission.28 In semiconducting materials, all excited electrons, independent of initial energy, relax to the band edge before fluorescence proceeds. However, Shang et al. demonstrated, like ours, constant emission maximum in the excitation range of 350–450 nm and red shifts in emission only at higher excitation wavelengths greater than 450 nm (Fig. S6, ESI†) due to the presence of multiple chromophores/fluorophores involved with aromatic and oxidation groups in the graphene oxide system.17 Furthermore, Kozawa et al.29 showed that there could be two emissions with excitations at 220 and 330 nm, which were attributed to the presence of different functional moieties such as hydroxybenzoic acid and dicarboxybenzoic acid. Therefore, violation of Kasha’s rule in the graphene oxide system as claimed by Cushing et al. appears to be controversial and debatable. The emission spectra of rGO and rGO-SO3H are shown in Fig. 3. Interestingly, the emission peak appears at 358 nm for both rGO and rGO-SO3H upon excitation at 240–270 nm. This shows that the sulphonation of reduced graphene oxide does not alter the emission profile. The observed results suggest that the origin of fluorescence in rGO remains unaltered with the introduction of the phenyl sulphonic acid group. However, sulphonated graphene produced from rGO-SO3H through reduction with hydrazine hydrate shows the emission maximum at 430 nm with an excitation of 310–340 nm. Therefore, the observed results clearly demonstrate that the reduction of oxygenated groups generates different fluorescence centers. It may be assumed that the reduction of oxygen containing groups may facilitate the extended delocalization of p-electrons involving the phenyl sulphonate group, which leads to the red-shift of fluorescence emission in SG to 430 nm from 358 nm of rGO-SO3H. We have followed the pH dependence of the fluorescence of GO and SG. The pH of the solution was appropriately adjusted with 0.1 M HCl or 0.1 M NaOH. We observed that the variation of pH in the range 4–9 had no influence on the spectral nature. However, Kundu et al.22 showed the change in the fluorescence spectra with pH. In our opinion, the main drawback in their experimental design was that pH of the solution was
Fig. 3
PL spectra of r-GO and rGO-SO3H (inset) with different excitations.
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Fig. 4 PL spectra of SG with different excitations in water (pH 6.5) and inset: PL spectra of SG dissolved in commercial buffers of pH 4 and pH 9.2.
maintained with commercially available buffer capsules of respective pH. We have checked the fluorescence of commercial buffer capsules and found that these capsules display fluorescence at the similar position to that reported in the respective pH. The emission spectrum of SG in water at pH 6.5 is shown in Fig. 4. The PL spectra of GO and SG recorded at pH 4 and 9.2 in the wavelength range of 350–550 nm under excitation at 320 nm (maintained with buffer capsules) are also presented (inset). For SG in pH 4 the emission maximum occurs at around 435 nm, which is also the emission peak position of buffer. We observe that appropriate care should be taken when emission of SG is followed. Excitation with 250 nm can be a problem as a strong emission appears at 350 nm, since we identify that this peak is primarily due to phenyl sulphonic acid used for sulphonation.30 The emission spectra of blank sulphanilic acid are presented in the inset of Fig. 5. Thus, to overcome this interference, in obtaining pure emission of SG, excitation wavelengths 310–340 nm should be used, as there is no absorption of pure sulphanilic acid in this
Fig. 5 PL spectra of reaction mixture excited at different wavelengths in the absence of graphene materials. Inset: Emission of sulphanilic acid with different excitations.
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Scheme 1
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Reaction products of hydrazine hydrate with sulphanilic acid.
range. Therefore, with this excitation, the emission peak appearing around 430 nm (Fig. 4) is exclusively that of the emission spectrum of sulphonated graphene.16 Furthermore, we followed the possible reaction products of hydrazine hydrate with sulphanilic acid (Scheme 1) in the absence of GO under the similar experimental conditions. Surprisingly, we find that there are two emission peaks at 350 nm and 450 nm in the reaction mixture (Fig. 5). It is apparent that the peaks at 350 and 450 nm are due to unreacted phenyl sulphonic acid and sulphonated phenyl hydrazine, produced respectively. However, this product being highly water soluble is removed by proper washing of the synthesized SG and thus, ensures no interference in spectral measurements of sulphonated graphene.
4. Conclusions In conclusion, the synthesis of water soluble graphene and accurate determination of its fluorescence characteristics are of utmost importance for the use of graphene based materials in various bio-medical applications, such as sensors, imaging devices, drugcarriers, etc. Water soluble sulphonated graphene and intermediate compounds/materials formed upon sulfonation of graphene oxide were analyzed through spectroscopic techniques. The present report reveals that violation of Kasha’s rule and pH dependence on spectral features of graphene materials as claimed recently are controversial and debatable, which may have serious ramifications in the growing applications of the graphene-based materials. We have also pointed out that proper care should be taken in monitoring the fluorescence of sulphonated graphene in view of possible interference from the components produced during sulphonation.
Acknowledgements The authors wish to thank IIT Kharagpur for providing the TEM facility. One of the authors (S. Maiti) is thankful to DST, Govt. of India, for the award of the INSPIRE fellowship. Two of the authors (S. Kundu and S. Mondal) are thankful to the UGC, Govt. of India for a NET-Junior Research Fellowship. Another author (D. Ghosh) is thankful to CSIR, Govt. of India for a Senior Research Fellowship.
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