In this study, we demonstrate separation of analytes in attoliter volumes using a single CNT. We utilized fluorescent dyes as analytes and fluorescence microscopy for detection owing to high sensitivity of the technique and small volumes, transport rates and length scales involved in the experiments. We employed DPE, which is a non-polar solvent that has a high solubility for the dyes and easily wets the CNTs. We covered the drops of non-polar solvents and the nanotube between them with a larger water drop to suppress heat induced evaporation. High polarity of water prevents wetting of the outer nanotube surface by DPE and causes the DPE drop to assume a non-wetting (contact angle >90°) shape minimizing energetically unfavorable surface interactions. Thus, when two drops are connected to two ends of a CNT, fluid transport from one drop to the other occurs exclusively through the CNT channel. However, using this set of liquids does not prevent outer surface wetting when graphitic CNTs are used (Fig. S4), probably because of enhanced favorable interactions between graphitic walls of CNTs and non-polar organic solvents. Meanwhile polar and hydrophilic solvents can also be used for separation by modifying the setup as explained in Supplementary Information section 1.6, however we persisted with non-polar solvents in this study as they provide a convenient means for demonstrating the concept. Our setup (explained in the methods section) overcomes two key issues critical for success of experiments involving femto-liter (or smaller) samples: rapid evaporation of liquids with moderate to high vapor pressures at ambient conditions and the outer surface wetting of the CNT. When two drops (that of pure DPE and dye mixture) are contacted with the two ends of a CNT, a Laplace pressure difference may generate fluid flow25 between the drops owing to small uncontrollable variations in the sizes and shapes of the drops. However this pressure gradient is insignificant in our experiments and its direction varies in individual trials, dampening out the variance with an increased number of experiments. Furthermore, we did not observe any change in the droplet size over the course of any experiment, suggesting that fluid flow was negligible.

Both fluorescent dyes (green and red) are soluble in DPE but are transported through the CNT channel at different rates corresponding to their diffusion coefficients and interactions with CNT walls. This initially results in a high ([green]/[red]) concentration ratio (Fig. 1 (c)) recorded from the pure DPE drop owing to the high diffusion coefficient (as shown later) and lower retention of the green dye by the CNT walls. The concentration ratio then decays to the equilibrium value of 1.6±0.9 and the relative transport is solely determined by relative diffusion rates due to the occupation of all active sites on the inner CNT surface by dye molecules.

To increase the CNT-dye interactions and enhance the separation, the CNT's internal surface area should be increased, which can be achieved by packing the tube with nanoparticles. However we have not yet been able to achieve a liquid flow rate desirable for fluorescence detection through a tube packed with iron oxide or diamond nanoparticles30. Therefore we used CNTs with smaller multiwalled CNTs grown inside (Fig. S1(b) and Supplementary Information 1.2)27,28. The altered initial part of the [green]/[red] vs time curve observed in Fig. 1(c) can be attributed to the interaction of the inner MWCNTs with the dye molecules. In this case, as suggested by our experimental data, the interaction with the green dye molecules is stronger than with the red dye molecules, resulting in an initial retention of the green dye. In all cases, we expect the eventual [Green]/[Red] value in the pure solvent drop to converge to ∼0.7, equaling that in the original solution if the experiment is allowed to run for sufficiently long times.

Liquid-liquid extraction was demonstrated by employing a pure solvent that selectively dissolves one solute as schematically represented in Fig. 2 (a). Thus when pure 1-OD is used as a solvent to prefill the CNT and a mixture of the green dye and Nile Red in DPE is contacted at the other end, the green dye (which is soluble in both solvents) can be selectively extracted into 1-OD phase, leaving behind Nile Red (which is weakly soluble in 1-OD) in the DPE phase (Fig. 2(b)). Indeed, similar transfer rates of the green dye were recorded from both pure DPE or 1-OD droplet (Fig. 2 (c)(i)) while negligible Nile Red transport was detected in 1-OD compared to that in DPE (Fig. 2(c)(ii)).

Owing to uncertainty in the operating temperature and viscosity due to intermittent laser exposure, we modeled the diffusion process using a finite element method software. The initial slope of the concentration-time curves has been calculated as a function of diffusion coefficient of the solute and CNT length using finite element analysis and plotted in Fig. 3 using which diffusion coefficients of solutes in solvents can be determined under experimental conditions used in our study. Further, Fig. 3 can assist in selecting the CNT length for separation of molecules with different diffusion coefficients. Depending on the minimal detectable concentration change of the analyzed compound, different tube lengths may be needed. A longer and thinner nanotube is favorable for better separation due to higher aspect ratio31,32, however it will also reduce the flow rate due to a higher viscous force making it hard to detect at low concentrations. CNTs used in this study so far were 190 nm in I.D. to enable easier and more accurate detection. However, tubes with diameters down to 10 nm can be produced by the template-assisted growth in anodized aluminum oxide (AAO) membranes.

The diaphragm-cell model, which was developed for diffusive transport across a uniformly thick diaphragm, is also useful in determining diffusion coefficients of solutes used in our setup. While the model ignores tortuosity of the diaphragm pores and convective transport due to buoyancy, the CNTs in our studies are synthesized as straight and uniform channels and tortuosity is no longer an issue. Further, buoyancy effects are negligible at the length scale of our experimental setup. Moreover, as shown in FEA simulations in Fig. S5, the concentration profile across the length of a nanotube becomes linear in less than 10 seconds even for a low diffusion coefficient solute. Therefore the diaphragm-cell model (which also assumes a linear concentration profile) is expected to provide results with high accuracy for our experiments. As can be seen in Table S2, the results obtained from both FEA simulations and the diaphragm-cell model closely match each other.

The observation of clearly separated green and red fronts inside the CNT (Fig. 4 (b)) during capillary filling further establishes the important point that the CNT walls interact differently with distinct chemical species, indicating that CNTs can be used as a chromatographic column. In these experiments we used mineral oil as the mobile phase since the diffusion of the red dye in the oil is slow (taking more than 90 minutes) and easier to record due to a higher oil viscosity. Further, it is not required to submerge the system in water owing to negligible evaporative loss of mineral oil (low vapor pressure as shown in Table S1). Several methods to functionalize CNTs have been developed in the past, which makes individual CNTs highly versatile for performing chromatographic separation. In the present work, we coated the CNTs with paramagnetic iron oxide nanoparticles30 and examined capillary filling of the dye mixture. Herein we observed the propagation speed of the red front to be much slower than through empty CNTs (Fig. 4c). This reduced propagation speed must be due to interactions between the diffusing red dye molecules and the iron oxide nano-particles, since the CNT column gets filled by capillary action within seconds, leaving diffusion to be the only mechanism for further movement of red dye through the CNT column. Therefore we fitted the two sets of data points (plain CNTs and Iron Oxide coated CNTs) with quadratic curves which are typical of diffusive transport and a close fit was observed.

In the separation techniques presented, the ability to detect a small number of transported molecules will be the limiting factor rather than CNT diameter, while attempting to push the limits of the techniques to smaller length scales or in employing more dilute solutions. Surface enhanced Raman spectroscopy (SERS) or electrochemical detection may prove beneficial in these applications as SERS allows single molecule sensitivity33 and electrochemical detection is routinely used to study transport of single protein molecules through nanoholes34. We used individual CNTs on a substrate for demonstration purposes. For developing functional CNT-based chromatographic columns, these tubes can be assembled into nanofluidic devices35 or used as nanopipette tips10 allowing separation and analysis of attoliters of fluids in forensics, nanoanalytical chemistry and single-cell analysis.

Overall, miniaturization of separation processes is highly desirable, but comes with many challenges. In this study we developed a method to overcome sample evaporation and eliminate wetting issues to perform attoliter volume separation in liquid phase. We showed that the experimental setup utilizing single (190 nm I.D., 200 nm O.D.) nanotube (nanopipe) could be used to measure diffusion coefficients of solutes on small length scales. The walls of an individual carbon nanotube could be chemically functionalized and used as stationary phase in liquid phase separation. Separation of two fluorescent dyes with non-overlapping excitation and emission wavelengths was demonstrated. The selectivity of separation could be controlled by packing the nanotubes with nanopartcles and we demonstrated this by growing multi-walled CNTs inside a larger CNT and by coating CNTs with iron oxide nanoparticles. CNTs can also be packed with nanoparticles to create nanoscale packed chromatographic columns24,30. Further, by using a solvent that selectively dissolves one dye, we demonstrated the process of liquid-liquid extraction. Thus we were successful in demonstrating various separation techniques with the smallest individual separation column ever reported, which holds great promise for minimally-invasive single cell analysis.