Table 1.

Selected techniques for GFN characterization and quantification

Method	Overview	Strengths	Limitations
Spectroscopic
Absorbance 8, 32, 40, 44, 92, 96, 113, 114, 116, 230	Measures absorbance of aqueous sample; can include ultraviolet, visible, or near infrared wavelengths; absorbance can be related to mass concentration using the Beer-Lambert law; with analytical ultracentrifugation (AUC), different fragment sizes of material can be measured with absorbance	Except for AUC, absorbance spectrophotometers are readily available in many environmental laboratories	Interference from other sample components; relatively high detection limit; only applicable for aqueous samples; controlled GFN dispersion quality required
Raman 113, 136, 137, 145, 159, 160, 231–233	Measures G, D and G’ vibrational bands in dry powder, polymer nanocomposites, and tissues	Minimal sample preparation; enables GFN characterization; compatible with in vitro and in vivo samples; can be used with a microscope; low detection limits achieved using resonance Raman conditions	Some matrices may produce interferences; sensitive to laser power; requires calibration or a reference peak for quantitative analysis; background fluorescence can interfere; samples dispersed in an organic solvent are less common but this is possible
Fluorescence 103	Measures fluorescence emission intensity after excitation of GFN at a known adsorption band	Available in many environmental laboratories; fluorimetry is highly sensitive; rapid technique	Interference of other fluorescent materials (e.g. polymer or environmental matrix); non-specificity of GFN signal; only applicable for aqueous samples; controlled GFN dispersion quality required; may work better for graphene oxide (GO) versus graphene because GO is more highly fluorescent
X-ray Photoelectron Spectroscopy (XPS)234–236	Measures the atomic surface concentration of carbon (top 10 nm) and can provide some information on oxidation state; relative concentration of GFN can be determined in a dry matrix if matrix has a very different conductivity relative to the GFN	Provides atomic information and oxidation state of GFN	Requires dry down and a high vacuum environment; doesn’t distinguish nanomaterial carbon from background carbon unless charging occurs and background material identity is known
Spectrometric
Inorganic Elemental Analysis of Metal Coordination to GFN Functional Groups 141, 155	Measures divalent metal cations coordinated to GO functional groups	Multi-elemental capability and extreme sensitivity of ICP-MS allow for an accurate and selective determination of metal content coordinated to GFN in a wide range of matrices at ngL−1 or sub ngL−1 levels, the rapid sample throughput of this method is attractive for routine screening; potential for covalent attachment of metals rather than coordination to minimize desorption of the metal tags during measurements	Carbon is generally not detectable with standard ICP-MS methods; metal release from carboxyl groups using strong acid is required prior to analysis; other carboxyl groups in environmental samples can interfere; carboxyl group content can vary between different GFNs; divalent metal cations can dissociate from carboxyl groups since they are not covalently attached; divalent metal cations can increase agglomeration state in water samples; will not work for pristine graphene since it does not contain functional groups
Microscopic
Atomic Force Microscopy (AFM) 32, 113, 115, 237–241	Measures the surface features of a sample by dragging or tapping a cantilever over the sample; the dimensions of identifiable GFN particles can be determined by the movements of the cantilever	A reliable technique for determining sheet thickness and lateral dimensions	Deposition bias, measurement bias, and detection errors are all possible in most samples
Hyperspectral Imaging 156, 242–247	Measures reflectance (or absorbance and transmittance) spectra of GFNs in a darkfield (visual near infrared /short-wave infrared spectral range) mode using a high-power halogen light source, resulting in 2D-optical images with full spectral information (400 nm to 1000 nm or 900 nm to 1700 nm, respectively) in each pixel (a pixel can be as small as 128 nm) 248	Easy sample preparation, provides optical and spectral information, allows spatial localization of particles without the need for labelling, can provide semi-quantitative information	Spectral mixing in complex samples/composites, long analysis times, spatial resolution may not be sufficient to differentiate individual small-sized GFNs from their aggregates (especially when stacked), which might impact quantification. Relatively expensive instrumentation.
Scanning Electron Microscopy (SEM) 137, 158, 159, 237, 247, 249	Measures the interaction of a finely collimated electron beam with the GFNs; secondary electrons emitted by atoms excited by the electron beam can be used for image formation	Provides 3-D morphological properties of GFNs; GFNs may be identifiable in complex matrices based on morphological criteria	Labor-intensive, often only qualitative information
Transmission Electron Microscopy (TEM) and Scanning Transmission Electron Microscopy (STEM) 112, 113, 157, 159, 160, 178, 237, 244, 250	A TEM passes a parallel beam of electrons through a selected sample area and detects the transmitted electrons that pass through the samples. The main difference with the STEM mode is that it scans very finely focused beam of electrons over the sample selected area in a raster pattern.	Provides morphological properties of GFNs; GFNs can be identified in energy filtered TEM images	Challenging sample preparation for tissues; it may be very hard to detect GFNs in complex samples at low concentrations
Laser Scanning Confocal Microscopy 110, 159, 178, 179	Uses a laser to excite fluorophores from a fluorescent marker tagged to GFNs or optically detects reflected light. The technique generates a series of focused image planes in the z direction by scanning with point illumination suppressing out-of-focus signal using a pinhole in front of the detector; three dimensional images are generated by combining the series of focused image planes.	Relatively easy technique for tracking translocation of GFNs in biological tissues	Only qualitative, or at best, semi-quantitative. Fluorescence probes may photo-bleach, and may be cytotoxic or interfere with normal biological processes. Reflection mode may be unable to distinguish GFN from other materials in the matrix that scatter light similarly.
Transient Absorption Microscopy 133, 251–254	A typical pump–probe technique whereby a modulated pump field (typically a pulsed laser) excites the electrons in the sample. A probe (another light source) then interacts with the photoexcited sample to obtain an absorption spectrum	Relatively fast, highly sensitive, and label-free technique that can be used to visualize GFNs in living cells and live animals. May provide quantitative data in well-dispersed GFNs	Light-absorbing matrices may introduce strong background signals. GFNs may have to be functionalized to improve their dispersability for quantitative analyses
Thermal
Thermal Gravimetric Analysis (TGA) 173–176	Quantification of mass percentage of phases with distinct thermal stabilities under a variety of reactive gases (usually inert or air) and relatively rapid temperature programs (e.g., heating rates of 5 °C/min to 20 °C/min; room temperature to ca. 950 °C); each sample takes 1 h to 2 h total; a systematic shift in the TGA profile as a function of GFN loading can potentially be measured since GFNs can enhance the thermal stability of materials	A rapid technique that allows for the quantification of multiple phases in a single sample; good for complex matrices; no special sample preparation needed	Effect of thermal ramp rate and reactive atmospheres on apparent phase distribution is not well understood (and is largely ignored), detection limits are relatively high for solid matrices since only small masses can be analyzed, potential for interferences between sample matrix (e.g., polymer, other carbon nanomaterials, soot, or black carbon) and GFN decomposition temperatures; good GFN dispersion quality required for systematic TGA profile shift; drying required
Differential Scanning Calorimetry (DSC) 173	Measures the thermal transitions of materials relative to a reference pan. The relative energy required or released is measured as a material is heated or cooled through a thermal transition; this technique has been used to measure the shift in the glass transition temperature (Tg) as a function of GO loading	A rapid technique and good for complex matrices; no special sample preparation needed	Thermal ramp rate can affect the transition temperatures; detection limits are relatively high for solid matrices; good GFN dispersion quality required for systematic DSC profile shift; dry-down required; might only be useful for samples containing polymer
Total Organic Carbon (TOC) Analysis 255–257	TOC analysis can be conducted on water or soil samples by oxidizing (chemical, heated catalyst, UV) carbon to carbon monoxide or dioxide which is detected by infrared or other types of detectors	TOC analysis has been used successfully with CNTs and fullerenes and once with few layer graphene (FLG) to investigate binding of NOM to FLG	Very little optimization of temperature or catalytic conditions have been examined; its application to CNT stock solutions have been consistent with prepared masses; any organics, such as natural organic matter, in solution or soils will interfere; this is a non-specific method and thus matrices that contain sufficiently high concentrations of other carbon nanomaterials (e.g., graphene), soot, or black carbons would impact the technique; with the more common instrument setups (680 °C maximum temperature), the temperature used is not sufficiently high to combust the FLG but would most likely be high enough for GO to combust
Programmed Thermal Analysis (PTA) 96, 170, 211, 258	While the temperature is ramped, there are two phases: inert followed by oxidizing for measuring organic and elemental carbon, respectively. Detects carbon by having evolved organic carbon be converted to CO2, then converted back to methane, and analyzed using a flame ionization detector. If organic carbon is converted to elemental carbon during the inert phase, there is a correction that can be performed.	Very reliable technique for detecting elemental carbon in environmental matrices, this technique could differentiate between types of GFNs based on their thermal stability; there is an ability to quantify mass	Too much organic carbon in a sample causes peak overlapping between elemental and organic carbon which affects the accuracy; similar carbonaceous materials such as CNTs and fullerenes will be counted in the GFN peak if they exist in the sample; unless the peak from GFN is far enough from the peaks for other carbonaceous material, it is difficult to exclude the other carbonaceous materials, however, adjusting the temperature program might help to some extent; GO does not separate from matrix unless a strong reducing agent is used followed by extraction prior to sample analysis
Isotopic Labeling
Carbon-14 Labeling 37, 42, 57, 106, 154	Can be used to quantify carbon-14 labeled GFNs following combustion in a biological oxidizer or direct addition to a scintillation cocktail; measures beta emissions using liquid scintillation counting (LSC); autoradiography can provide spatial distribution of radioactivity	Provides definitive quantification of GFNs in complex matrices; can be used as an orthogonal technique to develop other analytical techniques; can be used to identify degradation products and GFN quantities in tissues or released from polymer nanocomposites	High cost to synthesize radioactively labeled GFN; safety concerns; limited availability of radioactively labeled GFN; C-14 not inherently part of GFN that would be released into the environment
Additional Techniques
Gravimetric 259, 260	GFN mass concentration in air is estimated by determining total particle number (e.g. during GFN production) while accounting for background particle concentration. In suspensions, GFN concentration is estimated by drying a fraction of the suspension and weighing it, or by determining the fraction of GFNs not suspended by weighing the mass of GFN particles settled at the bottom of the container	Uses readily available equipment except in airborne measurements which require special instrumentation	Limited to high GFN concentrations, except in airborne measurements where the sensitivity of equipment may be reasonably high. The technique is nonspecific, and thus only applicable in relatively simple systems/matrices