Table 2.

Selected techniques for CNT Quantitation

Method	Overview	Strengths	Limitations
Spectroscopic
Absorbance65,109,150,151	Measures absorbance of aqueous sample; can include ultraviolet, visible, or near infrared wavelengths	Readily available in many environmental laboratories	Interference from other sample components, relatively high detection limit, only applicable for aqueous samples
Near infrared fluorescence (NIRF) 15,29,31,152	A specific emission spectra can be used as an identification tool of SWCNTs; the intensity of the fluorescence signal can be used for quantification of SWCNTs	Quantification/Detection at very low limits of detection	Limited to non-functionalized SWCNTs; semi-conducting SWCNTs but not metallic SWCNTs can be detected
Raman13,122,123,153–164	Measures radial breathing (SWCNT), G, D and G′ vibrational bands in dry and various solvent suspended samples, tissues	Minimal sample preparation, enables CNT 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 for quantitative analysis
Spectrometric
Inorganic Element Analysis29,32	Measures trace catalytic metallic elemental impurities intercalated in the CNT structure (Cr, Co, Cu, Fe, Mo, Ni, Y, Zn), analysis of bulk metal content; the applicability of this approach could be impacted by removal of the metal catalysts by purification but catalysts located within the CNTs often remain after purification processes	Multi-elemental capability and extreme sensitivity of ICP-MS allow an accurate and selective determination of metal impurities of CNT 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	Carbon is generally not detectable with standard ICP-MS methods, quantitative sample dissolution is required prior to analysis; incomplete sample digestion, release of metal ions from the CNTs in the sample matrix, or elemental contamination from the sample digestion steps could lead to an important bias in the bulk metal content determination; the feasibility of using this technique could depend partly on if the metal contents of the CNTs are known a priori
Single particle inductively coupled plasma-mass spectrometry (spICP-MS)22	Metal catalyst impurities are used as proxies to detect and quantify CNTs; the applicability of this approach could be impacted by removal of the metal catalysts by purification but catalysts located within the CNTs often remain after purification processes	Potential capability for the size, size distribution, and particle number concentration determination of CNT; high selectivity to differentiate CNT at extremely low concentrations from naturally occurring carbon- containing species (i.e. cells, organic detritus, humic acid); very low detection limit	Size/length estimation requires the invalid assumption that metal content is homogeneous among the CNTs, very small particles cannot be separated from the background, leaching of catalysts in the sample matrix prior to spICP-MS analysis can bias the result, only applicable for aqueous samples; the feasibility of using this technique could depend partly on if the metal contents of the CNTs are known a priori
Microscopic
Atomic Force Microscopy109,165	Measure the surface features of a sample by dragging a cantilever over the sample; the length of identifiable tubes can be determined by the movements of the cantilever	Most trusted technique for determining number and length	Deposition bias, measurement bias, and detection errors are all possible in most samples
Hyperspectral Imaging166,167	Measures reflectance spectra of NPs in a darkfield (visual near infrared/short-wave infrared spectral range), resulting in 2D-optical images with full spectral information that contain a full spectrum (400 nm to 1000 nm or 900 nm to 1700 nm, respectively) in each pixel; CNTs appear bright against a dark background	Easy sample preparation, provides optical (i.e. differentiation between single nanotube and nanotube- agglomerate) and spectral information, allows spatial localization of particles, can provide semi-quantitative information, short-wave infrared spectral range could be applicable for detection of SWCNTs	Currently long analysis times, visual near infrared not specific for CNTs, many potential analysis artifacts
Photoacoustic (PA)24,168–170	PA measures the acoustic response to the rapid volume change resulting from the absorption of an optical pump beam and the transfer of heat to the surrounding environment	Suitable for detection in liquids such as water and complex media such as plants, minimal sample preparation, can be quantifiable, excellent penetration depth enables samples > 100 μm, works equally well with metallic and semiconducting SWCNTs and MWCNTs, label free, unaffected by some complex media issues including carbon-on-carbon	Signal is dependent on absorption and heat transfer to material surrounding the CNTs, can be 10x lower sensitivity than PT, medium surrounding CNTs must be transparent to the beams, heating laser must overlap with absorbance of the CNTs, signal scales with size of CNT cluster, non-transparent media may cause detection issues, quantification may require diameter and length distributions
Photothermal (PT)24,168,169	PT measures the optical scattering response of a probe beam to the change in local environment refractive index that results from the absorption of an optical pump beam and the transfer of heat to the surrounding environment	Suitable for detection in liquids such as water and complex media such as plants, minimal sample preparation, can be quantifiable, penetration depth can handle samples up to 10 μm, works equally well with metallic and semiconducting SWCNTs and MWCNTs, label free, unaffected by some complex media issues including carbon-on-carbon, sensitivity down to single particle sensitivity, lower LOD than absorbance-based measurements	Same as Photoacoustic plus is limited to thin samples (< 100 μm)
Scanning Electron Microscopy and Scanning Transmission Electron Microscopy	Measures the interaction of a finely focused electron beam with the CNTs; secondary electrons, and transmitted electrons can be used for image formation	Provides detailed morphological properties (length, width, shape) of individual CNTs; individual CNTs can be localized in complex matrices based on morphological criteria	Labor intensive, often only qualitative information
Transmission Electron Microscopy (TEM)27,66	Illuminates a selected sample area (parallel electron beam) and detects the transmitted electron after passing through the samples	Provides detailed morphological properties (length, width, shape) of individual CNTs; high resolution can be used to distinguish between SWCNTs and MWCNTs; CNTs can be identified in energy filtered TEM images	Challenging sample preparation for tissues; it may be very hard to detect NPs in complex samples at low concentrations; low contrast (conventional TEM) due to reduced interactions between CNTs at the electron beam at high acceleration voltages
Thermal
CTO-37518	Quantification of carbon that remains after combustion at 375 °C for 24 h under excess air sample and subsequent chemical oxidation	Particularly good for complex matrices such as soil and sediment	Not fully tested for suspensions, requires high concentrations of CNTs and low concentrations of interferences (e.g., soot interfering with MWCNTs or graphene with SWCNTs)
Thermal Gravimetric Analysis (TGA) 20,171,172	Quantification of mass percentage of phases with distinct thermal stabilities under a variety of reactive atmospheres (usually air) and relatively rapid temperature programs (e.g., heating rates of 5 C/min to 20 C/min,; room temperature- ca. 950 °C); each sample takes 1 h to 2 h total	A rapid technique that allows 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, potential for interferences between sample matrix (e.g., other carbon nanomaterials, soot, or black carbon) and CNT decomposition temperatures
Thermal Gravimetric Analysis-Mass Spectrometry (TGA-MS) 14	TGA coupled with mass spectrometric detection of evolved gas fragments, typically in the 2 to 300 m/z range	Mass fragments can give insight into the chemical structure of the source material (e.g., C/H/O ratios or unique evolved fragments)	Current mass spectrometers have poor mass resolution (ca. 1 amu), relatively high detection limits, and low sampling rates relative to the chamber flush rate (i.e., consequently, only a small portion of the evolved mass is transferred to the MS); all reduce identification accuracy and increase detection limit
Total Organic Carbon (TOC) Analysis71	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 detectors	TOC analysis of waters has been used to measure CNTs in stock solutions in water	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 would 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
Thermal Optical Transmittance (TOT) 16,23	As the sample is analyzed under programmed temperature, the volatilized and combusted carbon travels to an oxidizing oven, where it is transformed into carbon dioxide (CO2); the amount of elemental carbon is determined based on the CH4 signal measured using a flame ionization detector; sample is first heated under inert conditions to remove volatile organic carbon, then oxidizing carrier gas is used for elemental carbon; the portion of TC that is organic carbon or elemental carbon is defined by the method, which determines where the organic carbon-elemental carbon split is placed post-analysis; this split can be automatic on the basis of automatic optical correction; the optical transmittance or reflectance is observed throughout analysis, and the split is placed where the transmittance/ reflectance returns to the initial reading; for samples in which optical correction does not work, a manual split defined by the analyst should be used	Very reliable technique for detecting elemental carbon in environmental matrices, this technique could differentiate between types of CNTs based on their thermal stability	Too much organic carbon in a sample causes peak overlapping between elemental and organic carbon which affects the accuracy; similar carbonaceous materials such as graphene and fullerene will be counted in the CNT peak if they exist in the sample; unless the peak from CNT is far enough from other carbonaceous material, it is difficult to exclude the other carbonaceous materials but adjusting the temperature program might help to some extent
Isotopic labeling
Carbon-13 Labelling21,32,78	A measure of the ratio of 13C to 12C, applicable for all CNTs but works best for isotopically enriched or depleted CNTs	Instrumentation is readily available in many environmental laboratories	Highly dependent on matrix and large variability may be observed for CNTs that are not specifically 13C enriched
Carbon-14 Labelling15,26,30,99,124,152,173–181	Measures beta emissions from carbon-14 emissions, can be used to quantify liquids after mixing with scintillation cocktail or any matrix after combustion in a biological oxidizer, autoradiography can provide spatial distribution of radioactivity	Provides definitive quantification of CNTs in complex matrices, can be used as an orthogonal technique to develop other analytical techniques, can be used to identify degradation products	High cost to synthesize radioactively labeled CNTs, safety concerns, limited availability of radioactively labeled CNTs
Other radioactive isotopes96–98	Measures release of emissions from a radioactive isotope that is associated (e.g., attached to a polymer wrapping the CNT) with the CNT	This approach can enable extremely low detection limits, can be used with a range of CNT surface functionalizations, non-destructive sample is possible for gamma emitters	Artifacts are possible if the radioactive isotope becomes separated from the CNT, it may be challenging or impossible to determine if this occurred in complex matrices without orthogonal CNT quantitation techniques
Additional Techniques
Analytical Ultracentrifugation (AUC) 60,165,182–184	Measurement of sedimentation velocity distribution, can be used to determine particle density or size/shape distribution	Can measure entire CNT population via absorbance or interference measurement, high resolution, little size bias	Finicky technique that requires well understood and controlled samples for robust analysis
Gravimetric185	The CNT concentration in suspension is estimated by drying a fraction of the suspension and weighing it, or by determining the fraction of CNTs not suspended during the dispersion process (e.g., by sonication) by weighing the mass of CNT particles at the bottom of the container	Uses readily available equipment	Limited to high CNT concentrations, only applicable for aqueous suspensions
Microwave Method62,125,186,187	Measures the temperature rise of a sample at a specific microwave energy within a specific timeframe	Straightforward method for CNT detection and quantification in biological tissue, low cost	Not commercially available; it still remains to be investigated for environmental samples if interferences arise from other carbon allotropes with similar behavior in the microwave field (e.g., carbon black, soot)188
aF4-MALS46	Measures a shape factor (ρ=radius of gyration/hydrodynamic radius) of particles present in a complex liquid sample (e.g. surface water, leachate, soil and sediment extract), which is indicative of the particle aspect ratio; comparing these results to a CNT-free sample can then be used for CNT detection	Allows for CNT detection in water, soils, and sediments; may be useful in exposure studies	Need for the baseline of a CNT-free sample, full quantitative use currently not straightforward, often low CNT recoveries for aF4

Abbreviations: Asymmetric flow field flow fractionation with multi-angle light scattering (aF4-MALS), analytical ultracentrifugation (AUC), carbon nanotube (CNT), chemothermal oxidation at 375 °C (CTO-375), inductively coupled plasma-mass spectrometry (ICP-MS), near infrared fluorescence (NIRF), multiwall carbon nanotube (MWCNT), photoacoustic (PA), photothermal (PT), single particle inductively coupled plasma-mass spectrometry (spICP-MS), single-wall carbon nanotube (SWCNT), transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), thermal gravimetric analysis-mass spectrometry (TGA-MS), total organic carbon (TOC), thermal optical transmittance (TOT).