Improving the adsorption capacity of graphene oxide. Effect of Ca 2+ on tetracycline retention

Tetracyclines (TCs) constitute a group of antibiotics that are commonly used to treat bacterial diseases, in veterinary medicine and as an additive in animal feed. This broad application has led to their accumulation in food products and the environment because sewage treatment plants cannot completely remove them. Therefore, the aim of this study was to synthesize graphene oxide (GO) and evaluate its TC adsorption properties in aqueous media. The effects of pH (between 2.5 and 11) and Ca 2+ concentration (between 0 and 1M) were thoroughly investigated. Structural, textural, and electrokinetic properties of the prepared GO were determined by N 2 adsorption/desorption, XRD, TEM, UV-vis, FTIR, XPS, thermogravimetry and electrophoretic mobility measurements. TC adsorption on GO is an interplay between the two main roles played by Ca 2+ : competitor or bridging cation. At low pH, there is cation exchange, and Ca 2+ behaves as a competitor of the positively charged TC species, decreasing adsorption as calcium concentration increases. At high, the formation of Ca bridges between the surface and TC (GO-Ca 2+ -TC) is favored, increasing the adsorption of the antibiotic by increasing calcium concentration. Different combinations of Ca 2+ and pH effects are important to improve the use of GO either as a pH-dependent and reversible TC adsorbent for decontamination or as pH-independent adsorbent for TC quanti�cation with electrochemical sensors.


Introduction
Tetracyclines (TCs) constitute a group of antibiotics, some natural and others obtained by semisynthesis, that cover a wide spectrum of antimicrobial activity.In addition to urinary tract infections, chlamydia, and acne, TCs can also be used to treat other bacterial infections such as respiratory tract infections, skin, soft tissue infections, among others (Castellanos et al., 2021; Ikonomidis et al., 2016;Mustapha et al., 2020;Pradier et al., 2018).The wide use of these antibiotics in veterinary medicine and also as an additive in animal feed, have brought as a consequence its presence in food products such as meat, milk, honey and chicken (Chopra and Roberts, 2001; Dasenaki and Thomaidis, 2015;Pérez-Rodríguez et al., 2018).TCs is not limited to food; they can also enter the environment because sewage treatment plants cannot completely remove them (Kumar et al., 2019).Moreover, there is a growing environmental concern about antibiotics because their presence in soils and waters leads to the emergence of resistant species.High concentrations of TCs and three degradation products were reported in the e uents (5.28-8.32 µg L -1 ) and sludges (34.6-49.6 µg kg -1 ) of three municipal wastewater treatment plants located in Turkey (Topal et al., 2016) and in aquatic organisms (4.23 to 208.14 ng g -1 ) (Han et al., 2020).Given these concerns, the development of analytical methodologies that allow the determination of low concentrations of tetracycline in environmental matrices is crucial.Among the analytical methods reported in different works, HPLC, ELISA, capillary electrophoresis, spectrophotometry, chemiluminescence and electrochemical approaches stand out (Abdulghani et Sattayasamitsathit et al., 2007).The main disadvantages of these techniques are that they have tedious sample pretreatment processes and long analysis times (Liu et al., 2018).On the other hand, electrochemical sensors are an attractive alternative for the detection of TCs due to their high selectivity, rapid detection and possibility of in situ applications (Liu et al., 2018).Graphene materials, as constituent of electrochemical sensors, seem to have adequate properties for these applications.Nanomaterials composed of graphene oxide (GO) have high surface area and high speci c capacitance due to its unique structure, which allows it to store a large amount of electrical charge.In addition, GO has oxygenated functional groups, such as epoxide (C-O-C), hydroxyl (C-OH), and carboxyl (COOH) groups, which are located both in the basal planes and at the edges of the material (Chen et al., 2012;Roy et al., 2011).These characteristics make them appropriate for the adsorption of different classes of compounds and therefore, for applications such as electrochemical sensors and biosensors for the determination, for example, pesticides, drugs, nitrogenous bases and others.(Gao et 2015) fabricated a carbon paste electrode by combining multiwalled carbon nanotubes and graphene oxide for TC detection.They applied adsorption separation differential pulse voltammetry (AdSDPV) in which tetracycline was rstly electroaccumulated and then analyzed.This technique only detects those analytes that are strongly adsorbed on the electrode surface.The sensor was applied to different samples such as arti cial urine, river water and pharmaceutical samples.Lorenzetti et al. (2020), on the other hand, applied a modi cation of AdSDPV to determine TC in milk and river samples using disposable screen-printed electrodes.The technique allowed analysts to improve the selectivity of electrochemical sensors by exploiting the adsorption properties of the sensing surface (Paleček and Bartošík, 2012).
Within the tetracycline's family, the so-called tetracycline (TC) is one of the substances that constitute this group of antibiotics.It has different acid groups in its structure and can exist under different ionic species and conformations depending on the pH.The presence of such groups in the TC molecule generates potential sites of interaction with metal ions and surfaces (Parolo et al., 2013).
The adsorption of TC on montmorillonite, mesoporous silice, graphene-based materials and others adsorbents have been previously studied (Brigante et al., 2014;Gao et al., 2012;Guo et al., 2020;Minale et al., 2020;Parolo et al., 2012a; Rivera-Utrilla et al., 2013).Nevertheless, there is little information on the TC adsorption in the presence of a divalent cation such as Ca 2+ .This cation is ubiquitous in natural media and is implicated in many biological and environmental processes.There are studies regarding its in uence on the adsorption of different organic and inorganic compounds on different adsorbents (Antelo et al., 2015;Li et al., 2013;Lin et al., 2017Lin et al., , 2018;;Stachowicz et al., 2008).Chowdhury et al., (2015) studied the in uence of pH, ionic strength, ion valence and the presence of natural organic matter (NOM) on the aggregation and stability of graphene oxide (GO) and three reduced GOs (rGO).They found that the stability depended on pH, ion valence, and concentration of surface functional groups.In the presence of divalent cations (Ca 2+ , Mg 2+ ), the increase in pH decreased the stability of GO, which was due to Ca 2+ adsorption on the surface functional groups of GO.In the presence NOM and divalent cations (Ca 2+ , Mg 2+ ), GO aggregates settled from suspension because Ca 2+ ions act as bridges between GO functional group and NOM.This indicates that pH and divalent cations can play complex roles in the adsorption properties of rGO and GO and could affect signi cantly adsorption and detection of third molecules such as tetracyclines.The present work focuses on obtaining GO nanoparticles with TC adsorption properties for a future application as electrochemical sensors or as TC sorbents for decontamination.The synthesis and structural characterization of GO is rstly presented, followed by an evaluation of the TC adsorption properties.Special attention is paid to the effects of Ca 2+ on the adsorption, which could be used to optimize the performance of GO as a sensor or as a TC adsorbent.

Materials
Graphite powder (Gr) with a particle size < 50 µm was supplied by Sigma-Aldrich.KMnO 4 (99.5%),H 2 SO 4 (95-98%), H 3 PO 4 (85%) and HCl (36-38%) were supplied by Cicarelli.Hydrogen peroxide (30% v/v) was supplied by Anedra and ethanol (96%) was supplied by Porta.Tetracycline hydrochloride (TC, purity 99%) was obtained from Parafarm and was used without further puri cation.TC stock solutions were prepared just before use to avoid degradation caused by oxygen and light.CaCl 2 and 0.01 M KCl solutions were used for Ca 2+ effect experiments and as electrolyte for zeta potential measurements.NaOH and HCl solutions were used for pH adjustment.
1.2 Synthesis of graphene oxide GO was synthesized using Tours' method (Marcano et al., 2010).A 9:1 mixture of H 2 SO 4 :H 3 PO 4 (360:40 mL) was added to Gr powder (3.0 g) and KMnO 4 (18.0 g), producing a slightly exothermic reaction, which increased the temperature of the reaction vessel to 35-40°C.The product of the reaction was then heated to 50°C and stirred for 12 h, cooled at room temperature and poured afterwards onto ice (~ 400 mL) with 30% H 2 O 2 (3 mL).This last procedure produced important bubbling, a yellow coloration on the product, which nally became brownish.The resulting mixture was centrifuged (10000 rpm for 20 min), and the supernatant was discarded.The remaining solid material was washed rst with 200 mL water, then with 200 mL 30% HCl, and nally with two washings of 200 mL ethanol.The washed material was dried overnight at 42 ºC in air.The obtained brown material will be referred as GO hereafter.

Characterization techniques
Textural characterization of Gr and GO were conducted with N 2 adsorption/desorption isotherms at 77 K using a Micromeritic Gemini V2.0 2380 equipment.Samples were degassed at 323 K for approximately 12 h.Surface area values were calculated from the linear adjustment of the Brunauer-Emmett-Teller (BET) equation with adsorption data obtained in the relative pressure (p/p•) range between 0.01 and 0.33.The total pore volume and the average pore size were obtained by the Barrett-Joyner-Halenda (BJH) method.X-ray diffraction (XRD) patterns were obtained with a Rigaku D-Max II-C diffractometer, with CuKα radiation (λ = 1.54 Å) of 40 kV and 20 mA.Scans were recorded between 5 and 60 º 2θ, with a step size of 0.02 º 2θ and a scanning rate of 2 º min − 1 .Crystal dimensions were calculated using the Scherrer equation.The morphology of GO was characterized using transmission electron microscope (TEM, JEOL 100 CX) images.The TEM micrographs of the GO sample were collected at a maximum accelerating voltage of 100 kV.The sample was prepared by dispersing a small amount of GO in ethanol via sonication for about 15 min.A few drops of this suspension were placed in 200 mesh grids provided with a Formvar lm.FT-IR spectra were obtained with a IRTracer-100 Shimadzu spectrophotometer.The sample was prepared in KBr pellets with 0.3% w/w of GO or Gr.The spectra were recorded between 400 and 4000 cm − 1 with a 4 cm − 1 resolution and a 32 min − 1 acquisition rate.X-ray Photoelectron Spectra (XPS) were obtained with a Thermo Scienti c K-Alpha + X-ray Photoelectron Spectrometer.Spectra were recorded at room temperature, using Al-K (1200W) radiation for excitation and a 180° double focus hemispherical analyzer in a vacuum chamber of 1-10 mbar.Spectra were collected for oxygen (O1s), carbon (C1s) and a survey to identify the different species.The deconvolution of the spectra was obtained by means of Igor software using Voigt functions.Thermogravimetric analyses (TGA) data were recorded on a Mettler Toledo TGA/DSC1 instrument, the samples were heated from room temperature to 1000 ºC (10 ºC min -1 ) in N 2 atmosphere.UV-Vis spectra were collected in the range of 200-700 nm using a Genesis 10S UV-Vis Spectrophotometer (Thermo Scenti c).A zetasizer (Nano-ZS) Malvern equipment was used to determine the zeta potential of Gr and GO particles at different pH values.Gr and GO suspensions were prepared in 0.01M KCl solutions, the pH was adjusted to the desired value and after 15 min equilibration, the electrophoretic mobility was measured.Zeta potential was calculated using the Smoluchowski equation.The effects of Ca 2+ on the electrophoretic mobility of GO at different pH values were also evaluated.For this, a stable GO dispersion was prepared in 0.01M KCl as electrolyte and in presence of different CaCl 2 concentration (0.001M and 0.01 M).The pH was modi ed gradually in all cases, and after equilibration the mobility was measured.

Adsorption studies
Adsorption studies under equilibrium conditions were carried out to investigate the removal capacity of tetracycline (TC) and the effects of pH and Ca 2+ on it.Batch adsorption experiments were carried out in 10 mL glass asks continuously shaken with an orbital shaker (200 rpm) at 22 ± 1 º C in duplicate.The concentration of TC in the equilibrium solution was quanti ed by UV-Vis spectrophotometry.Since the wavelength of the absorption maximum of TC changes with pH and presence of Ca 2+ , calibration curves in the concentration range of 0-48 mg L − 1 were performed at the corresponding pH values, with and without 0.01M CaCl 2 .
The equilibrium adsorption capacity (Q e , mg g − 1 ) of GO was calculated as Where c i and c e are the initial and equilibrium TC concentrations, V is the volume and m the mass of adsorbent used in the experiments.The ratio m/V is known as the adsorbent dose.Adsorption isotherms at 22°C were performed in 10 mL capped glass vessels varying TC initial concentration from 20 to 90 mg L − 1 with the same adsorbent dose (250 mg L -1 ).Two pH values were investigated (5 and 9.5).Once equilibrated, the dispersions were ltered with 0.45 µm lter, and the supernatant separated for TC quanti cation.Similar adsorption experiments were performed in the presence of 0.01M Ca 2+ , in order to evaluate the effects of calcium in the adsorption behavior of GO.

Effect of pH
The effect of pH on TC adsorption was investigated in the pH range 2.5-11.0.The initial concentration of TC was 30 mg L − 1 , and the adsorbent dose was 250 mg L − 1 .Capped glass vessels with a total volume of 10 mL were used.The pH effect was also analyzed in the presence of 10 − 2 and 10 − 3 M Ca 2+ .

Effect of Ca 2+
Additional experiments to evaluate the effect of Ca 2+ on TC adsorption were performed at constant pH and varying calcium concentration.The initial concentration of TC was 30 mg L − 1 , the adsorbent dose was 250 mg L − 1 and the nal volume was 10 mL.Ca 2+ concentration was varied from 0.0 to 1.0 M at two different pH values, 2.5 and 9.5.

Characterization
The results obtained by N 2 adsorption/desorption experiments are listed in Table S.1, in the Supporting Information.Gr has a BET area of 14 m 2 g -1 , which increased to 30 m 2 g -1 after the oxidation process.The average pore size of GO is 5.6 nm, indicating that it is a mesoporous material.In comparison with others graphene oxide obtained from natural graphite and oxidized by a modi ed Hummers method (Li  S.1, in the Supporting Information) also indicate that GO exhibits strong acidity while Gr is almost neutral (slightly acidic).
The XRD patterns and structural parameters of Gr and GO are shown in Fig. 1A and 1B, and listed in Table S.2 in the Supporting Information, respectively.Gr shows three main re ections around 26.6°, 44.7°, and 54.8° 2θ corresponding to (002), (101), and (004) crystallographic planes, respectively.The intense and sharp peak of the (002) plane suggests a highly ordered material, with multiple graphene layers (Mendoza-Duarte et al., 2020).After the oxidation, the peak at 26.6° 2θ disappeared as a consequence of the complete oxidation of Gr (He and Fang, 2016) and a typical peak associated to GO ((001) plane) appeared at around 9.26º 2θ.These ndings are indicative of a signi cant reduction of the close-packed hexagonal structure and an increase of stacking disorder due to the existence of intercalated oxygenous groups (Zhao et al., 2014).The increase in the interlayer spacing (from 0.34 nm to 0.95 nm) was attributed to the conversion of sp 2 carbon to sp 3 carbon, evidencing GO oxidation and exfoliation (Romero et al., 2018;Szabó et al., 2005).
The crystallites height (Lc) decreased after oxidation from 21.49 nm in Gr to 12.06 nm in GO (Table S.2, in the Supporting Information).The layer separation in graphene oxide enhanced by oxygenated functionalities is also evidenced by TEM analysis (Fig. 1C).Dark areas indicate the thick stacking nanostructure of several graphene oxide sheets.More transparent areas indicate thinner lms of a few layers of GO resulting from exfoliation (Stobinski et al., 2014).As it was reported in the literature, the wrinkled morphology is attributed to the presence of a large number of functional groups such as hydroxyl and carboxyl groups on the edge, and carboxyl and epoxide groups in the inner part of GO (Das et al., 2020).
The FT-IR spectra of Gr and GO, displayed in Fig. 2A  Figure 3A shows the TGA of Gr and GO and Fig. 3B shows the DSC of GO, all performed in N 2 atmosphere.Gr was stable in the whole range of temperature analyzed.On the contrary, GO begun to decompose at approximately 50ºC, and lost up to 85% of its total weight when heated to 200ºC.The rst endothermic peak is due to water desorption, with around 10% of mass loss.The higher mass loss occurred between 150-190°C, with two marked exothermic peaks at 162 and 181°C.This high thermal reactivity of GO can be attributed to the decomposition of the labile oxygen-containing moieties, promoted by the disruption of the multilayered stacks structure of GO (Gao et al., 2014; Hou et al., 2020).
The UV-vis spectrum of GO is shown in Fig. S.1, in the Supporting Information.It presented a characteristic absorption band at 230 nm and a broad shoulder at 300nm, which can be assigned to the π-π* transition of the C = C bonds and n-π* transition of the C = O bonds (carbonyl or carboxyl group), respectively.The presence of the shoulder at 300 nm is a good evidence of oxidation (Kartick et al., 2013;Li et al., 2017;Peng et al., 2015).On the contrary, Gr particles settled so quickly that only a clear supernatant remained, and that is what is observed in the UV-visible absorption spectrum.
The electrokinetic properties of Gr and GO are shown in Fig. 4A.Gr had a negative zeta potential of around − 22 mV at high and intermediate pH, and then the potential decreased as the pH decreased, reaching a nearly isoelectric point at pH 2.1.GO, on the contrary, exhibited a more negative zeta potential in the whole range of pH investigated.It only decreased from − 35 mV to -30 mV from pH 10.5 to pH 2.1.The negative zeta potential is in line with the fact that oxidation treatment introduced oxygen-containing functional groups on the surfaces of GO (Li et al., 2013;Li et al., 2003;Song et al., 2010).Consequently, GO suspensions resist more aggregation and tend to disperse better than Gr. Figure 4B shows that calcium produces signi cant changes to the electrokinetic behavior of GO.The zeta potential becomes less negative as calcium concentration increases, and the isoelectric point shifts to pH around 6 in 10 − 2 M calcium, evidencing interaction of Ca 2+ with the surface groups of GO.

Adsorption isotherms of TC on GO at different pH values and Ca 2+ concentration
TC adsorption isotherms on GO at pH 5 and 9 are presented in Fig. 5. TC adsorption decreased with increasing pH from 5 to 9.5 in absence of CaCl 2 .In the literature, the pH was found to impact similarly the adsorption of TC on different solids (Maged et al., 2020;Parolo et al., 2008;Topal and Arslan, 2020).The results suggest that electrostatics interactions are playing a role on the adsorption, because the adsorbent is negatively charged in the analyzed pH range (Fig. 4.A) and TC species increase their negative charge as pH increases (Parolo et al., 2008).In fact, at pH 5 TC is present in the zwitterionic (TCH ± ) and anionic (TCH − ) forms, whereas at pH 9.5 it is in its negative and doubly negative forms (TCH − and TC 2− ).Even though the net charge of TCH ± and TCH − is 0 and − 1, respectively, both species contain a positively charged group in their structure, the dimethylammonium group and thus, the molecules can arrange at the surface locating the positively charged group close to the surface and the negatively charged group(s) as far as possible from the surface (Parolo et al., 2012b), resulting in adsorption at pH 5.At pH 9.5, repulsive forces seem to prevail, and adsorption is nearly zero.These results were also corroborated by the pH effect adsorption assay (see below).
The effect of Ca 2+ on TC adsorption isotherms at pH 5 and 9.5 is also shown in Fig. 5.The isotherms practically did not change by changing the pH in the presence of calcium.At pH 5 the presence of calcium decreased the removal capacity of GO, whereas at pH 9.5 the divalent cation signi cantly increased the removal capacity.The result was that isotherms practically coincided at pH 5 and 9.5, when calcium was present.It is possible that at pH 5 Ca 2+ competes with the positively charged group of TC species, decreasing the adsorption.This competition is no longer operative at pH 9.5, where an opposite Ca 2+ effect was observed, probably as a consequence of the interaction of the divalent cation with negatively charged groups of the GO surface and negatively charge groups of TC species.Parolo et al., (2013) reported the same effects of calcium on TC adsorption on the negatively charged montmorillonite surface, postulating the formation of Ca 2+ bridges between the surface and TC.This seems to be also the case with GO, with Ca 2+ forming GO-Ca 2+ -TC bridges and thus inducing the adsorption of negatively charged TC species on a negatively charged surface.
The effect of calcium on TC adsorption was explored in more detail by investigating the removal at eight different pH values and at three different calcium concentrations.The results are shown in Fig. 6, and can be understood as an interplay between the two main roles played by calcium on TC adsorption, i.e., competitor or bridging cation.At pH lower than around 6, the competitor role of calcium prevails and thus TC removal decreased by increasing calcium concentration to 0.001 M or 0.01 M. At pH higher than around 6, on the contrary, calcium seems to act mainly as a bridge between the surface and TC, increasing TC removal by increasing calcium concentration to 0.001 M or 0.01 M. The general effect of calcium is, then, to atten the adsorption vs pH curves, making the removal only weakly dependent on pH.
The effects of calcium were further investigated by working at two extreme pH values (2.5 and 9.5), with six different calcium concentrations.The results are shown in Fig. 7, which presents TC removal at different Ca 2+ /TC ratios, for a constant TC concentration.At pH 2.5, where TC is fully in its cationic form, the results in Fig. 7 are typical of competition between calcium and TC for adsorption sites, with a monotonous decrease in TC adsorption as the Ca 2+ /TC ratio increases.At pH 9.5, instead, the trend is no so simple.TC removal increases by increasing calcium concentration up to a Ca 2+ /TC ratio of 11, passes through a maximum, and then decreases at higher Ca 2+ /TC ratios.Bridging calcium may be still responsible for the adsorption under these conditions.However, it is very di cult to completely understand the whole behavior, specially why the removal decreased at very high calcium concentrations.At such high Ca 2+ concentrations the surface probably becomes positively charged, as suggested by zeta potential data.In addition, the fact that the molar Ca 2+ concentration is 11000 times higher than that of TC may induce the formation of Ca 2+ -TC species with an excess of calcium in the structure and low a nity for the surface.In fact, speciation calculations by Parolo et al. (2012a) showed that at high calcium concentrations and pH > 6 the dominant species in solution in a Ca 2+ -TC mixture is Ca 2 TC 2+ , which may have low a nity for the positively charged GO surface.

Conclusions
GO nanoparticles were prepared and characterized for TC adsorption.Adsorption varies widely with pH and Ca 2+ concentration.In the absence of Ca 2+ , adsorption was high at low pH and decreased as pH increased.It can be seen that in the presence of Ca 2+ decreased TC adsorption at low pH but increased it at high pH.It is postulated that at least two different adsorption processes take place in the presence of Ca 2+ : at pH ≤ 5 there is cation exchange, and Ca 2+ behaves as a competitor of the positively charged TC species.At pH ≥ 5, the formation of Ca bridges (GO-Ca 2+ -TC) is favored, increasing the adsorption of the antibiotic.Consequently, in the presence of Ca 2+ , the pH does not produce signi cant variations in the adsorption of TC.The combination of Ca 2+ and pH effects are important to optimize the behavior of GO and the use of Ca 2+ or not will depend on the application needed.For example, not using calcium in the adsorbing media is important to reversibly adsorb TC at low pH and desorb it at high pH, which may be useful for decontamination and further regeneration of the adsorbent.Using Ca 2+ , on the contrary, may be very advantageous for using GO in an electrochemical sensor, which will have a pH-independent response, facilitating the use of the device.

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SUPPLEMENTARYMATERIAL.docx
et al., 2013), the studied sample has intermediate speci c surface area and pore volume (Bradder et al., 2011; Li et al., 2013; Srinivas et al., 2011; Sui et al., 2013).The pH values of Gr and GO dispersions (Table and 2B, show characteristic vibration bands of these materials.Gr spectrum resulted to be relatively at, with weak signals.The band at 1550 cm − 1 is attributed to the C = C vibration of graphene sheets.The spectrum of GO was very different, with stronger signals.The most intense band appears at 3400 cm − 1 and corresponds to the O-H stretching vibration of hydroxyl groups and water molecules.The deformation vibration mode of O-H groups appears at around 1410 cm − 1 .The band at 1740 cm − 1 was associated to the C = O stretching vibration of a carbonyl group; another band at approximately 1620 cm − 1 was attributed to the C = C skeletal vibration of the graphene sheets (unoxidized graphitic domains) (Saleh et al., 2017); a band appearing at 1220 cm − 1 was in turn attributed to the stretching vibration of epoxy C-O-C group; and the band at 1050 cm − 1 was nally assigned to the alkoxy C-O stretching vibration (carboxyl group) (Alazmi et al., 2016; Drewniak et al., 2016; Wang and Hu, 2011).A comparison of Gr and GO spectra clearly indicates that GO is the material with the highest proportion of oxygenated groups.The presence of oxygenated groups on GO was also detected by XPS.The elemental analysis obtained by XPS indicated C: 64.3 atom%, O: 32.1 atom%, N: 1.9 atom% and S: 1.6 atom%.The C/O ratio equal to 2 indicates a successful introduction of oxygen atoms into the ordered arrangement of graphite structure.The C1s XPS signal shows different types of carbon components.The C1s spectrum show four peaks that correspond to the following functional groups: carbon sp2 (C = C, 284.2 eV), carbon sp3 (C-C, 284.9 eV), epoxy/hydroxyls (C-O, 286.8 eV), and carbonyl (C = O, 288.6 eV) (Fig. 2.C) (Poh et al., 2012; Yu et al., 2016).The relative proportion of these groups is shown in Fig. 2D.

Figure 1 A 2 A 3 A
Figure 1