Kyle
Doudrick
*a,
Takayuki
Nosaka
b,
Pierre
Herckes
c and
Paul
Westerhoff
d
aDepartment of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. E-mail: kdoudric@nd.edu
bSchool for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ 85287-6106, USA
cDepartment of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA
dSchool of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, AZ 85287-5306, USA
First published on 3rd November 2014
Interest is growing for graphene as a nanomaterial for electronic and composite applications. Increased production and use of graphene warrants development of strategies to detect and monitor its effect on human health and the environment. A quantification method using programmed thermal analysis (PTA) was developed for few-layer graphene (FLG) and graphene oxide (GO). FLG exhibited strong thermal stability, which allowed for easy detection in matrices consisting of thermally weaker background organic carbon. GO (50% oxygen content) exhibited a weaker thermal stability than FLG, making quantification more challenging in the presence of thermally similar background organic carbon. To resolve this, an in situ reduction method using a reducing agent (sodium borohydride) was developed to remove surface-bound oxygen from GO. This was used in combination with a digestate (SolvableTM) to create an optimized extraction method for recovering FLG and GO from complex organic matrices. FLG and GO will enter sewer systems due to their use by industry and in consumer products. We investigated the applicability of this method for quantifying FLG and GO in wastewater biomass because they are likely to accumulate in wastewater biosolids, as these are commonly the first exposure route for novel materials in the environment. Spiking 20 μg of FLG and GO into a 200 mg dried biomass L−1 wastewater solution resulted in recoveries of 52 ± 8% and 80 ± 6%, respectively. Results from this study can be applied to the development of extraction methods for graphene from similar complex organic matrices (e.g., lung tissue, in vitro/in vivo studies, algae, daphnia) to support a range of human and ecotoxicological studies.
Nano impactWith increased graphene use in consumer products there is a growing concern over its effect on human health and the environment. Quantification methods are needed to understand the risk associated with graphene. In this study, we describe a method for quantifying graphene in complex organic matrices. This method is useful for monitoring graphene in the environment and determining its impact on human health. Given graphene's likelihood to end up in wastewater treatment plants, we demonstrate the applicability of this method for wastewater biosolids. The results presented in this study will also be fundamental for the further development of methods for quantifying graphene in other complex matrices (e.g., sediment, tissue). |
Detection methods such as X-ray diffraction3 and Raman spectroscopy4 are useful for characterizing graphene, but they do not allow for appropriate quantitative analysis. Thermogravimetric analysis (TGA) and ultraviolet–visible (UV–vis) spectroscopy can also be used to detect graphene; they are quantitative methods, although limited in that respect. TGA is useful for determining the thermal stability of graphene and can also quantify purity (i.e., metal content),5 but it is limited to purer, dry samples rather than graphene in complex environmental or biological matrices. UV–vis has been used previously to characterize the dispersion state of graphene oxide (GO) in aqueous solutions,6 and it can be used as a means of quantifying GO in aqueous solutions, but only if the dispersion (i.e., aggregation) state stays constant. The UV–vis sensitivity becomes poor for graphene (stacked sheets in aqueous matrix) and GO in aqueous solution below approximately 1.5 mg L−1 and 75 μg L−1, respectively (Fig. S1† showing UV–vis spectrum for graphene and GO). In more complex matrices (e.g., surface water), quantifying graphene and GO will be more difficult due to different aggregation states and matrix interferences in the same wavelength range, which is especially true for GO (peaks between 220–250 nm; Fig. S1†). The lack of analytical methods for quantifying graphene in complex matrices signifies a need to develop robust analytical strategies that include both quantification and sample preparation.
We have previously developed a quantification method for carbon nanotubes (CNT),7 and have applied it to CNTs that were extracted from lung tissue with a high recovery.8 This quantification method, termed programmed thermal analysis (PTA), is an organic carbon/elemental carbon analysis that determines carbon mass and separates CNTs from other forms of carbon on the basis of the CNT's thermal stability. This separation is achieved using a time-dependent temperature ramp program; thermally weaker carbon compounds (e.g., tissue, bacteria) evolve early in the program while thermally stronger carbon compounds (e.g., CNT, graphene) evolve later. The ability to separate distinct forms of carbon is important for avoiding background interferences when quantifying carbonaceous nanomaterials in complex matrices containing organic carbon.
Before PTA can be used to quantify CNTs in complex organic matrices, CNTs must be extracted to separate them from excess carbonaceous material that could interfere with the analysis. With proper extraction methods in place, CNTs can be concentrated and then quantified using a number of methods (e.g., TGA-mass spectrometry,9 gel electrophoresis,10 infrared,11 radio-labeling,12 microwave,13 UV–vis,14 and inductively coupled plasma-mass spectrometry15). Given the physical and chemical similarities between graphene and CNTs, we hypothesize that the same approach can be used for extracting and quantifying graphene.
For PTA, oxygen functional groups on CNTs are problematic because they complicate separation of CNTs from organic carbon during analysis.7 Graphene is expected to be easily amenable to PTA because of its low oxygen content and consequently high thermal stability.16 Alternatively, GO tends to have a very high oxygen content, with a carbon to oxygen ratio (C:O) on the order of 1:1; thus, its thermal behavior is similar to organic carbon. While the similar thermal behavior is not an issue for samples containing only GO (e.g., pure aqueous GO stock solutions), it interferes with analysis when quantifying GO in matrices containing organic carbon. GO can be transformed to “reduced graphene oxide” (RGO) using chemical reducing agents such as hydrazine17,18 or sodium borohydride.19–23 Removing oxygen makes graphene (oxide) more hydrophobic, which increases its tendency to aggregate and results in a more efficient separation and extraction. The key to any successful approach for environmental and biological samples will be doing this in situ (i.e., in a complex matrix) so that GO can easily be recovered.
With the increase in graphene production and the advent of new graphene-containing products, graphene is likely to enter into wastewater treatment plants. Given graphene's similarity to CNTs, it will presumably end up in wastewater effluent or wastewater biosolids (treated sewage sludge containing living/dead microbes and inert solids).24 Of these exposure routes, biosolids seem to be the most appropriate end-point for graphene and GO.25–28
The aims of this study were to (1) develop a PTA quantification method for graphene and GO and (2) develop a method for recovering graphene and GO from complex organic matrices. We utilized few-layer graphene (10–20 nm thick) in place of single-layer graphene due to the problem obtaining an aqueous solution of single-layer graphene. Because of the difficulty extracting oxygenated carbonaceous nanomaterials (e.g., GO) from complex matrices, we applied an in situ reduction method to increase hydrophobicity and improve recovery. Given the likelihood of graphene to end up in wastewater biosolids, we demonstrated an extraction and quantification method for wastewater biosolids to assist with fate and transport studies. The results stemming from this research can be leveraged to develop extraction methods for graphene from other biological matrices (e.g., lung tissue, in vitro/in vivo studies, algae, daphnia).
Sodium borohydride (99.99%, Sigma Aldrich, 480886), hydroiodic acid (57% in H2O, Sigma Aldrich, 210013), and ascorbic acid (reagent grade, Sigma Aldrich, A7506) were used as received. Solvable™ was obtained from Perkin Elmer. Solvable is a tissue solubilizer consisting of sodium hydroxide (≤2.5%), C10-16-alkyldimethyl, N-oxide (2–10%), and C11-15-secondary, ethoxylated alcohol (2.5–10%). Sodium hydroxide (97%, EMD SX0590), Tergitol 15-S-12™ (C12-14 secondary ethoxylated alcohol, CAS no. 84133-50-6, Dow Chemical Company), and N,N-dimethyldodecylamine, N-oxide (30% in H2O, Sigma Aldrich 40236) were obtained to examine the individual components of Solvable. Ultrapure water (18.2–18.3 MΩ cm) was used for all experiments.
The method detection limit (MDL) for GO or FLG in 1000 μg dried biomass was calculated using a t-distribution with 99% confidence (one tail, seven replicates, 5 μg graphene).29 The 95% lower (LCL) and upper (UCL) confidence intervals were calculated as 0.64 × MDL and 2.20 × MDL, respectively.29
XPS was used to investigate the C–C and C–O/CO bond content of GO. Fig. 2 shows the XPS analysis for GO in water and GO after treatment with NaBH4 in water (i.e., RGO). Two peaks were present, one at 284 eV, which is attributed to C–C/CC, and the other at 286–290 eV, which coincides to a number of carbon and oxygen functionalities (mainly C–O and CO). The C–O/C–C ratio for GO was 1.1:1, which agrees with the manufacturer's carbon to oxygen ratio of 1:1. The C–O/C–C ratio for RGO was 5.6:1, an approximate 5-fold decrease in the number of C–O/CO bonds. GO reduction also shifted the C–O peak to lower binding energies, indicating a change in the type of carbon–oxygen functionalities that remained on the GO. These results provide clear evidence that carbon–oxygen functionalities were removed by NaBH4 treatment. SEM images show that reduction of GO (Fig. S2a†) to RGO (Fig. S2b†) did not significantly alter the particle shape or x–y size (e.g., both large, 5–10 μm, and small (e.g., right side of Fig. S2b†), <1 μm, sheets were present), and stacked, plate-like structures were formed. Fig. 3 shows the PTA thermogram for RGO after treatment with 2% NaBH4 in water. Chemical reduction improved the thermal stability (i.e., peak shift to the right), providing additional evidence that oxygen functionalities were removed.
Fig. 2 XPS analysis of (a) GO and (b) RGO. The average position for CC and C–C was 284.0 eV, and the average position for CO and C–O was 287.0 eV and 288.6 eV for GO and RGO, respectively. |
Raman spectroscopy is used to determine the defect density of CNTs and graphene,4 defined as the ratio between the D-band (1350 cm−1) and G-band (1580 cm−1) (ID/IG). The defect density is an indication of the thermal stability7 and the degree of oxidation.30 We hypothesized NaBH4 reduction would decrease the defect density and result in an increase in the GO thermal stability due to a decrease in the number of oxygen functionalities. However, Raman results revealed that the ID/IG did not change significantly (>5%) after NaBH4 treatment. Although reduction of oxygen functionalities occurred (i.e., XPS and thermal stability results), the chemical reduction treatment did not heal defects. NaBH4 is known to reduce aldehydes and ketones into alcohols, and it is capable of reducing lactone and carbonyl groups to hydroxyl groups on functionalized CNTs.31 So, in the case of NaBH4 reduction of GO, presumably the GO functionalities are only being reduced as far as C–OH and C–H, and NaBH4 is not able to heal defects through C–C sp2 bond formation.
In water, NaBH4 enabled aggregation of GO, presumably a result of removing oxygenated functional groups, but separation via centrifugation was difficult (i.e., Fig. S5†). In a clean, aqueous matrix (i.e., only water and GO), filtration directly onto a quartz-fiber filter (QFF) is an option for separating the GO (e.g., 10–20 μm X–Y dimensions), but this is not an option for complex matrices because the filter will also collect interfering carbon compounds. For applications involving clean matrices free of carbon interferences, filtration may be an option; though retention using the QFFs, which are designed to function as air filters, may be poor for GO as observed for functionalized CNTs.7 Furthermore, if a different quantification method (e.g., electrophoresis, UV–vis) is used, the sample would need to be in a concentrated aqueous or powder form and not adhered to a filter.
In a Solvable matrix, which is the reagent used to solubilize organics (e.g., wastewater biomass (this paper), tissue8), GO aggregated and formed a very stable, compact pellet upon centrifugation. This is likely due to a combination of a high pH, double-layer compression from increased ionic strength, and the presence of two surfactants, which may cause a cloud-point like effect.32 The known individual components of Solvable were examined to determine the root of the effect. Both surfactants (10% concentration) alone and in combination caused aggregation while sodium hydroxide was not effective. Upon addition of NaBH4 to the surfactants, samples exhibited severe effervescence due to hydrogen generation, and GO was not easily recovered as it adhered to the vials, overflowed the vials along with the bubbles, or would not centrifuge into a pellet. However, adding sodium hydroxide to the two surfactants (individual or combined) curbed the effervescence. Therefore, the excellent performance of Solvable for extracting graphene can be attributed to a synergistic action of its components rather than a single species. Fig. 4 shows the percent recovery of RGO as a function of increasing NaBH4 concentration. Recovery with Solvable alone (i.e., no reducing agent) was 75 ± 0.5%. Adding low concentrations of NaBH4 (e.g., 0.04%) did not show improvement with an average recovery of 76 ± 3%. Increasing the NaBH4 concentration to 0.4% resulted in a slightly higher recovery (81 ± 3%), but recovery over 90% wasn't achieved until greater than 2% NaBH4 was used (95 ± 5%), with a maximum recovery of 97 ± 0.4% observed using 8% NaBH4. The improved physical recovery was attributed to a reduction in oxygen content, resulting in increased aggregation of the graphene particles. Removal of carbon–oxygen bonds shifts the hydrophilic nature of graphene to be more hydrophobic, resulting in improved aggregation during centrifugation. Reduction also decreases the amount graphene that would otherwise be lost in the organic carbon PTA background (i.e., during the inert phase as shown in Fig. 1).
Fig. 4 Percent recovery of RGO after GO reduction using various concentrations of NaBH4 (0, 0.04, 0.4, 2, 8%). Error bars indicate one standard deviation (each direction) for triplicate samples. |
When using PTA for quantifying graphene, the thermal stability (i.e., peak oxidizing temperature) is important for separating graphene from background organic carbon during analysis. Fig. 5 shows PTA mass loss curves under oxidizing conditions for GO using different extraction conditions. Surfactants have been previously shown to reduce the thermal stability of CNTs,7 and we observed the same effect for GO treated with Solvable, an alkali reagent containing surfactants. Solvable decreased the thermal stability of GO significantly, with an onset approximately 130 s earlier and 50 °C lower. Reduction of GO in water with NaBH4 improved the thermal stability (Fig. 3), so we hypothesized that this would improve the GO stability after Solvable treatment. Using low concentrations of NaBH4 (e.g., 0.04%) after the Solvable treatment only increased the thermal stability slightly (~20 s), but using a higher concentration of NaBH4 (>2%) returned the thermal stability close to the original (Fig. S6†). The improvement in the thermal stability may account for the improved recovery when using greater than 2% NaBH4 (i.e., Fig. 4). To achieve optimal extraction, a combination of Solvable and at least 2% NaBH4 is recommended.
Fig. 5 Mass loss curves for GO (~20 μg) under oxidizing PTA conditions using different extraction conditions. “Sol” is Solvable and “BH” is NaBH4. |
Without Solvable treatment, FLG and GO detection in biosolids was not possible because the amount of biomass collected in the pellet overwhelmed PTA and resulted in indistinguishable peaks for graphene and biomass. Using the extraction method of Solvable and 2% NaBH4, GO/RGO and FLG (20 μg) recoveries from 1 mg dried biomass (0.02 μg graphene μg−1 dried biomass) were 80 ± 6% and 52 ± 8%, respectively. Although FLG is easier than GO/RGO to detect in a complex matrix using PTA because it is more thermally stable, physical separation from the biomass using centrifugation was less efficient, resulting in a lower recovery. We observed that FLG was very stable in Solvable (before and after biomass treatment), with little recovery occurring via centrifugation (<5%). Although FLG is already in a “reduced” form, adding NaBH4 helped to improve the FLG aggregation and extraction. We also examined nitric acid as a digesting agent in place of Solvable to determine if pH or surfactants were an issue. Like Solvable, FLG was more stable in nitric acid (pH < 0) than in ultrapure water (pH = 5.6), likely due to increased surface charge separation, but extraction was worse than with Solvable. This agrees with previous results showing Solvable to be optimal over common agents (e.g., nitric acid, hydrochloric acid, hydrofluoric acid, etc.) used for extracting CNTs from rat lung tissue.8
Solvable was efficient at dissolving the biomass, but a small amount of background carbon still remained and interfered with GO/RGO peaks (Fig. 6); no interference was observed for FLG. The interference for GO/RGO was consistent across triplicate samples with an average of 2.2 ± 1 μg. When the amount of biomass was increased to 5 mg (1 g biomass L−1), GO/RGO peaks were indistinguishable due to the false positive from interfering background carbon that remained after treatment. To improve the extraction for GO/RGO from high concentrations of biomass, we developed a phase-separation method by extending the heating time of the NaBH4 step to 36 h. This causes the water and surfactant phases of Solvable to separate (Fig. 7a), and after centrifugation, RGO remains mostly in the top surfactant phase (Fig. 7b). Similarly, when done in a wastewater matrix (i.e., 1 g biomass L−1), the undigested (interfering) biomass transfers into the water phase, and the RGO remains in the surfactant phase (Fig. 7c). This results in a physical separation of the RGO and the interfering background carbon, allowing for easy recovery of the RGO only. Note, control samples digested with Solvable for 36 h (i.e., no NaBH4) did not show any significant (<5%) additional removal of biomass interference. Therefore, using NaBH4 to separate the biomass and RGO into different phases is key for improving recovery in wastewater with a high biomass concentration. Using the phase-separation method, the recovery of RGO (20 μg) from 5 mg biomass was 110% ± 13%. Recovery greater than 100% is attributed to undigested biomass constituents interacting with RGO, causing the biomass to remain in the surfactant phase. This interaction is presumed to be adsorption of the biomass to RGO as no interfering background carbon from the biomass was observed in the surfactant phase for triplicate control samples that did not contain RGO. The phase-separation was not successful for FLG as the majority of the FLG transferred to the water phase along with the undigested biomass. The advantage of using the phase-separation method over the centrifugal separation method for GO is that larger amounts of biomass can be used while avoiding interferences from undigested biomass. However, in other instances, the centrifugal method is preferred because it is simpler, less time consuming, and useful for both graphene types. A detailed schematic of the two methods is shown in Fig. S7.†
Fig. 6 PTA thermogram showing biomass interference for GO in wastewater biosolids. Solvable and 2% NaBH4 treatment. |
Reported recoveries are ideal as they were obtained using a lab-grown, clean biomass. When using biosolids obtained from a wastewater treatment plant, the recovery values are expected to increase due to presence of soot particulates, which behave thermally similar to graphene, thereby creating a false positive.7 Similarly, the presence of carbonaceous nanomaterials (e.g., CNTs, fullerenes) in environmental samples with graphene is possible,33 further complicating recovery when using PTA. While GO/RGO and FLG used herein can be separated and quantified using PTA (e.g., Fig. S7†), the presence of CNTs with a similar thermal stability as GO or FLG would be difficult to distinguish with PTA alone. Ideally, a graphene standard (similar to the NIST single-walled CNT standard reference material, SRM 2483) would be used to create a spike standard addition curve in order to quantify the amount of background soot (or CNTs) interfering with graphene. With the challenge of thermally similar carbonaceous materials present (e.g., soot) or predicted (e.g., CNT) in the environment, PTA and similar thermal methods alone are not currently suitable, and analytical advancements to these methods and more selective extraction methods are needed. However, PTA, in its early analytical development as described herein, is an excellent tool for monitoring the fate/transport and toxicity of graphene for model systems and organisms, respectively.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4en00134f |
This journal is © The Royal Society of Chemistry 2015 |