Density gradient ultracentrifugation for colloidal nanostructures separation and investigation

Accepted Manuscript Review Density gradient ultracentrifugation for colloidal nanostructures separation and investigation Pengsong Li, Anuj Kumar, Jun Ma, Yun Kuang, Liang Luo, Xiaoming Sun PII: DOI: Reference:

S2095-9273(18)30172-5 https://doi.org/10.1016/j.scib.2018.04.014 SCIB 385

To appear in:

Science Bulletin

Received Date: Revised Date: Accepted Date:

7 December 2017 27 March 2018 28 March 2018

Please cite this article as: P. Li, A. Kumar, J. Ma, Y. Kuang, L. Luo, X. Sun, Density gradient ultracentrifugation for colloidal nanostructures separation and investigation, Science Bulletin (2018), doi: https://doi.org/10.1016/j.scib. 2018.04.014

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Review Recived 07-December-2017;Revised 27-March-2018, Accpted 28March-2018 Density gradient ultracentrifugation for colloidal nanostructures separation and investigation Pengsong Li, Anuj Kumar, Jun Ma, Yun Kuang, Liang Luo*, Xiaoming Sun* State Key Laboratory of Chemical Resource Engineering, College of energy, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China E-mail: [email protected]; [email protected] Abstract In this article, we review the advancement in nanoseparation and concomitant purification of nanoparticles (NPs) by using density gradient ultracentrifugation technique (DGUC) and demonstrated by taking several typical examples. Study emphasizes the conceptual advances in classification, mechanism of DGUC and synthesis-structure-property relationships of NPs to provide the significant clue for the further synthesis optimization. Separation, concentration, and purification of NPs by DGUC can be achieved at the same time by introducing the water/oil interfaces into the separation chamber. We can develop an efficient method “lab in a tube” by introducing a reaction zone or an assembly zone in the gradient to find the surface reaction and assembly mechanism of NPs since the reaction time can be precisely controlled and the chemical environment change can be extremely fast. Finally, to achieve the best separation parameters for the colloidal systems, we gave the mathematical descriptions and computational optimized models as a new direction for making practicable and predictable DGUC separation method. Thus, it can be helpful for an efficient separation as well as for the synthesis optimization, assembly and surface reactions as a potential cornerstone for the future development in the nanotechnology and this review can be served as a plethora of advanced notes on the DGUC separation method.

Keywords: density gradient ultracentrifugation, isopycnic separation, rate zonal separation, colloidal nanostructure, nanoseparation.

1. Motivation of nanoseparation The discovery of size-dependent properties of nanomaterials, colloidal nanoparticles (NPs) with tunable size and shape has attracted vast attention due to their wide applications in catalysis, energy conversion, bio-issues, nanomedicines [17]. As well known, size and geometric control of nanomaterials are the important factors for the discovery of intrinsic size/shape dependent properties and bottom-up fabrication approaches of functional nanodevices [8-12], and known as “nano-size effects”. Monodisperse nanomaterials with unified morphology and size are ideal need, but most of the synthetic systems are often yielded as polydisperse nanostructures. The intrinsic non-uniformity in temperature and concentration field always exists, and the size deviation would inevitably exist in a one-pot synthesis. One solution is to control the size directly during the synthesis by applying very strict synthetic conditions [13-17], but this strategy only suits for the very limited systems, otherwise, in most cases, the products are only roughly “monodisperse” with certain deviations. The other way is to develop the nanoseparation technology as an advanced post-synthesis purification method. In what follows, we will discuss the progress of nanoseparation methods. 2. Nanoseparation methods for nanostructures The use of suitable techniques for the separation of nanostructures is necessary to achieve the desire monodispersity [18]. Currently, a number of separation techniques viz. electrophoresis, size exclusion chromatography, magnetic, membrane filtration and centrifugation are used to separate the various nanomaterials with varied shape and size. Especially the centrifugation method consists of ordinary centrifugation and DGUC. In this section, we will give a brief introduction of the above-mentioned separation techniques. 2.1 Electrophoresis The separation mechanism of electrophoresis relies upon the surface charges of nanostructured materials, which drive charged particulates to move toward the electrode of opposite charge under the external electric fields effect [19, 20]. The separation efficiency is strongly influenced by many factors, such as types of buffer solution, pH value, the concentration of buffer solution, ionic strength and the electric field intensity [21], usually by changing the surface charge density and thus the charge/mass ratio and consequently the movement speed of the NPs. Electrophoresis has been used for the separation of metal sulfides and metal nanoparticles [22]. The nanosheets, nanorods, and nanoparticles of Ag were purified in the latter case, in other words, enriched into various fractions, to show size and shape dependent plasmon resonance wavelength [23].

2.2 Chromatography Chromatography is a powerful tool for the separation of the components from a mixture based on differential partitioning between the mobile and stationary phases. Since nanoparticles can be regarded as “huge inorganic molecules” judging from the size and molecule weights, size exclusion chromatography (SEC), which is commonly used for the separation of macromolecules, turns out as one of the best choices [18, 24, 25]. Consequently, the materials (e.g., water-soluble CdS [26] and gold [24, 27, 28] nanoparticles) get varied retention time and thus can be separated by size/volume under the appropriate control. The advantage of chromatographic techniques is that the separation process can be rationally designed, and the compositions of the stationary phase and mobile phase can be turned to match with the nature of colloidal particles. However, the quantity of separated production can only be achieved in milligram level and the separated nanoparticles might get stuck in the pores of stationary phase in SEC case, or adhere on the surface in the IEC case, and be hard to elute, which could decrease the reproducibility and increase the cost. 2.3 Magnetic field The magnetic method was stabilized for the separation of magnetic nanomaterials based on the size-dependent magnetic properties [29, 30]. Yavuz et al. [31] demonstrated the separation of Fe3O4 nanoparticles with the diameter of 4, 6, 9, 12 and 20 nm, respectively, which built the base for separation. Differential magnetic catch and release (DMCR) method was thus developed [32], which was used to separate and purify magnetic materials, like, hybrid Au-Fe3O4 and FePt-Fe3O4 nanoparticles [33]. 2.4 Membrane filtration The membrane is a barrier with uniform pore distribution to realize the selective filtration using the organic or inorganic material. Membrane filtration is a membranebased method wherein pore size dictates the retention and elution of material from the sample. Membrane filtration has been used frequently for water purification or wastewater treatment, and recently extended to the separation of biological materials, or colloidal nanostructures as the pores are made in a nanoscale with uniform size [34, 35], that is showing the merits of its affordability, convenience, and versatility [36]. 2.5 Evaluation criteria on nanoseparation Besides the separation methods discussed above, there are still many methods that can be used to sort nanoparticles, including selective precipitation using anti-solvents, or centrifugation using density gradients. Which is the ideal nanoseparation method for the colloidal nanocrystals? What are the standards used to assess the separation methods? Following 6 considerations might be helpful for future researchers as

reference: (1) high efficiency to separate one sample into many as fractions with obvious difference; (2) high versatility towards different systems, for instance, twodimensional (2D) nanosheets, one-dimensional (1D) nanorods/nanowires, and zerodimensional (0D) nanodots; (3) wide range of feasibility for nanoparticles with wide size ranges (e.g.,1–500 nm) and colloidal nanoparticle systems with different solubility; (4) little sample loss. Try to avoid any possible “solid-solid” (sample-solid separation media) contact to minimize sample loss, or efficiency loss of the separation system; (5) the intrinsic properties of colloidal particles after separation should not be changed; even their ligands should be well protected while any possible aggregation of the colloids should be avoided; (6) the system for separation is reusable, or easy to duplicate and/or optimize. By parameters optimization in secondary (or repeated) separation, the separation efficiency can be further improved. Amongst the above separation methods, centrifugation methods are emerging due to its intrinsic advantages of high efficiency and relatively low cost and attracting the researchers’ wide attentions which have been discussed in detail as below. 3. Categories and principles of density gradient ultracentrifugation Centrifugation is a process which can utilize the centripetal forces to sedimentate the heterogeneous mixtures [37] and this method was developed into a variety of forms with the development of equipment [38]. And centrifuge separation technologies which can be divided into three types: differential separation, isopycnic separation, and rate zonal separation [39]. Differential centrifugation is also known as pelleting separation, which is a method of using a certain speed centrifugation to separate materials from homogeneous suspension, it has been widely applied to separate and purify nanoparticles by centrifuge-redispose circles [40–42]. Besides differential centrifugation, DGUC [43] has become the other main section of centrifugation and has been successfully applied in the separation and purification of various nanoparticles. This method has a wide range of applications involving nearly all kinds of materials including metal, metal oxides/sulphides, carbon materials, semiconductors, etc. As a general, nondestructive, and scalable separation method, DGUC has recently been demonstrated as an efficient way of sorting colloidal nanoparticles according to their differences in chemistry, structure, size and/or morphology [39, 44, 45]. As an effective complementary process to synthesis optimization [46], nanoseparation [33, 45, 47–50]is attracting more interests for obtaining strictly monodisperse NPs [36, 51]. Nowadays, the DGUC method [39, 52–54] has been demonstrated as a versatile method to obtain monodispersed colloidal NPs in both aqueous and organic phase. It could separate NPs according to sizes, densities, and morphologies [55]. 3.1 Differential separation Differential separation is a centrifugal method based on the different settling velocity of nanoparticles in a homogeneous media [56]. As shown in Fig. 1, the

technique is accomplished by repeatedly centrifuging a suspension from low speed to high speed to achieve separation. In a typical process, different sized nanoparticles move toward the bottom of centrifuge tube with different settling rates, and the mixture would be gradually separated into two parts: the large ones in the precipitate and the smaller ones in supernatant. Low centrifugation speed should be used first to precipitate the large particles to the bottom of the tube. After removing the precipitate, higher centrifugation speed should be used for the residual suspension, and mediumsized particles would precipitate. Finally, the precipitation of the smallest sized particles needs the highest centrifugation speed. The differential centrifugation has been widely used especially for the separation, extraction, enrichment of bioactive materials such as animal/plant virus and subcellular components etc. The obtained precipitated samples must go through the re-suspension, re-centrifugation, and re-washing processes to get much purer form of the particles. This method is suitable for the samples which contain big weight difference between the fragments, or the difference between sedimentation coefficients (the measurement on settlement, which is defined as the quotient of a particle’s sedimentation velocity over the acceleration) of mixed samples should be more than 10 times. For example, Chen et al. [57] used three different centrifugation speeds to separate graphene oxide (GO) nanosheets with different sizes.

Fig. 1 Schematic illustration of the typical differential separation method 3.2 Isopycnic separation Isopycnic separation has been demonstrated as a powerful tool for the separation of carbon nanotubes [53, 58]. With this method, when the density of particles suspended in the mixture suspension is different from that of the gradient medium, under centrifuge, they either settle or rise along with the gradient. The particles keep moving until they reach the positions where the medium density is equal to those of particles, which is the meaning of isopycnic (equal density) state [56]. In practical, long-term centrifugation (usually longer than 12 h) is required since the particles take time to find the same-density layer (ρmedia=ρparticle) to locate therein (Fig. 2a). Hence, the separation efficiency of the isopycnic separation depends on the density slope of gradient media along the centrifuge tube: shallower ones usually correspond to higher resolution. Fig. 2 Schematic illustration of typical isopycnic separation (a) and rate-zonal separation (b). The optimized separation states as shown in red boxes. Reprinted from Ref. [39], Copyright (2014), with permission from Elsevier.

Due to the high resolution of the linear density gradient, the isopycnic separation achieves the high separating capability to purify nanomaterials with 0.0032

g/cm3density difference [59]. To use the isopycnic method for separation, the net density of targeting particles (including solvation layer) should be ranged between those available for density gradient media. And the nanoparticles with net density in the range of roughly 0.9 to 1.4 g/cm3 can be separated using the isopycnic separation method, which is the common range of gradient media. Since the core materials usually possess bulk density (ρc) larger than 2 g/cm3 (for sulfides, oxides or carbon), it is even larger than 8 g/cm3 mostly for metals, while the density of aqueous phase gradient media is usually less than 1.4 g/cm3 (such as 60% sucrose solution) [60]. Thus, only those colloids with tiny enough size (e.g., < 2 nm) can get colloids with net density (ρp) less than 1.4 g/cm3, which excludes the isopycnic method for heavy (high density) or big (>2nm in thickness) particles separation. For example, the density of Au is 19.3 g/cm3, and the net density of Au nanoparticles with the surface ligand of oleylamine (hydration shell thickness is assumed to be 2 nm) can be calculated with the increase of nanoparticle size, as shown in Fig. S1 (online). 3.3 Rate zonal separation If isopycnic separation is regarded as a “static state” separation, rate zonal separation [61] should be considered as a “dynamic” separation method based on particle size, shape, and density. Fig. 2b shows the typical process of rate zonal separation. The method is to put the samples on top of the density gradient which has a smaller scale of density (ρmedia<ρparticle) and mildly changed slope. Mass and the viscous resistance are different for the particles with different shapes and sizes under a given centrifugal force, which leads to different sedimentation coefficients and consequent different particle sedimentation velocity, and effective separation. If the gradient density is lower than the minimum density of the separated particles, the particles would keep moving down to the centrifuge tube with the increasing time. Hence, rate zonal separation undertakes in a strong centrifugal field, costs short period of separation time (generally, not more than 4 h). Thus, parameters for the rate zonal separation, such as time, rate and gradient density, should be adjusted specifically for the target particles. The rate zonal separation method has been successfully applied to sort the various colloidal nanostructures, such as FeCo@C, Au nanoparticles, graphene, and CdS nanorods [62–64]. 3.4 Mathematics on sedimentation behavior of nanoparticles In the density gradient centrifugal system, the driving force of the particles’ movement is the centrifugal force (Fc) [63, 65], which is represented as Eq. (1): Fc=mω2x, (1) where m (g) is the mass of the particle, ω (rad/s) is the angular velocity of the rotor, x (cm) is the distance from the nanoparticle to the rotation center. In liquid media, the particle movement characteristics depend on the density, size and shape of a particle, centrifugal force, and viscosity of the liquid medium (reverse friction) etc. [62]. And other effects like gravity can be ignored here because the

centrifugal force is typically >1,000 times higher than it. The schematic diagram of density gradient in a centrifugation tube is shown in Fig. 3a. Fig. 3 Schematic illustration of separation mechanism. (a) Schematic diagram of density gradient centrifugation tube, ρ: the density of medium. (b) A hydrodynamic colloidal nanoparticle model. (c) Stress analysis of the nanoparticle in a centrifugal field. Reprinted with permission from Ref. [66], Copyright (2016) American Chemical Society.

In a density gradient liquid medium, each nanoparticle has a solvation layer on its surface [62, 66]. As shown in Fig. 3b, for ideal spherical particles with core density (ρc), radius (r), hydration shell thickness (h), and the hydration shell density (ρh), the net density (ρp) can be estimated by the Eq. (2):

 p   h   c   h  r 3 /  r  h  . 3

(2)

Fig. 3c shows the force analysis of the particle in the separation process and Eq. (3) describes the dynamics of the particle during the centrifugation under the collective influence of the centrifugal force (Fc), the reverse viscous resistance (Ff) and buoyancy (Fb) [67].

d2 x m 2  Fc  Fb  Ff , dt

(3)

where t (s) is the centrifugal time. Independent on separation systems, due to acceleration and deceleration for centrifuge, the particle movement can be divided into three stages: the first one is an accelerative process with alterable positive acceleration; the second one is uniform motion without acceleration and the last one is a decelerated stage with an alterable negative acceleration. During the centrifugation, when the particle achieves uniform motion [67], the force equilibrates (d2x/dt2=0), the dynamic Eq. (3) of the particle can be written as Eq. (4): Fc=Fb+Ff. (4) In the centrifugal field, the centrifugal force (Fc) is mω2x, and the accelerated speed isω2x. So, the buoyancy [68] of particle in centrifugal field can be described as Eq. (5):

Fb 

m

p

 m 2 x,

(5)

where, ρm is the density of gradient media, ρp is the density of nanoparticle. According to Stokes’ Law [69], when a spherical rigid particle with a radius (r) (insoluble in the liquid medium) move with a speed of dx/dt under the centrifugal force field, it can affect the reverse viscous resistance (Ff) of the solution according to Eq. (6):

Ff  6 r 

dx , dt

(6)

where η (Ns/m2) is the viscous coefficient of the liquid medium, r (cm) is the radius of the particle. From Eqs. (4)–(6), the sedimentation rate of the particle can be described as follows Eq. (7) by using the product of volume and density to replace the mass. 2 dx 2r (  p   m ) 2   x, dt 9 f

(7)

where, f is the shape factor of particle in the solution. f =1 for ideal spherical system and f >1 as the shape deviate from sphere shape. When the net density of particle equals to the density of gradient media, the particle would lose the driving force; as a result, it would stop and keep this state even prolonging the centrifugal time that is isopycnic separation, as shown in Fig. 2a. For a given centrifugal system, the density and viscosity of the liquid medium are known quantitatively and for a certain particle, r, ρp, η and f are also known quantitatively, “2r2(ρp–ρm)/9ηf” can be defined as sedimentation coefficient (s) and can be calculated as Eq. (8). Thus, sedimentation coefficient is the ratio of particle velocity to its acceleration. dx / dt (8) s . 2x

On the other hand, from Eq. (4), for ordinary nanoparticles, the sedimentation coefficient (s) [70] can be defined as Eq. (9): dx / dt (9) s  m[1  (  / )] / f . 2x

media

particle

Thus, there are mainly three factors that can affect the sedimentation coefficient (s). (1) Effect of mass (m); greater the mass of particle, greater the sedimentation coefficient (s), the particle with higher mass travels down the centrifuge tube rapidly. (2) Effect of shape of particle (f=fractional coefficient of particle); more spherical particle moves with high sedimentation speed because more spherical particle has lower fractional coefficient value. (3) Effect of ρmedia/ρparticle value; generally, ρmedia/ρparticle value decides the sign of sedimentation coefficient (s) and particles settling orientations during the centrifugation; (i) whenρmedia/ρparticle=1, the value of sedimentation coefficient (s) is equal to zero, particles locate in the certain position that mean ρmedia=ρparticle, (ii) when ρmedia/ρparticle<1, the value of sedimentation coefficient (s) is greater than zero, particles settle along the direction of centrifugal force that mean ρmedia<ρparticle, and (iii) when ρmedia/ρparticle>1, the value of sedimentation coefficient (s) is less than zero, the particles float against the direction of centrifugal force that mean ρmedia >ρparticle. If the density of particle is larger than the density of the gradient media, the Fc, Fb and Ff should be unbalanced (d2x/dt2≠0), the dynamics equation of the particle can be described as Eq. (10) [66]:

d2 x 9 dx  m   p 2    x  0. 2 2 dt 2  p ( r  h) dt p

(10)

It can be deduced from the above formula that the positions of the particles in the centrifugal tube after separation are determined by centrifugal time, centrifugal rotational speed, density gradient, and medium viscosity, etc. For a given separation system, we can get the optimized parameters by simulation before density gradient ultracentrifugation [66]. In addition, the physical quantity of shape factor (f) can be introduced to correct the effect of morphology on the dynamics equation [63]. 4. Applications on density gradient ultracentrifugation 4.1 Isopycnic separation in aqueous media DGUC has become a promising tool to purify the nanoparticles especially in aqueous media, because it is adapted to biological applications wherein a lot of molecules would keep activity only in aqueous media. In a given centrifugal field, when the density of suspended particles is different from the gradient media, they will start to move and, stay at the gradient layer with exactly the same density under a certain centrifugal force. DGUC was firstly utilized to isolate single-walled carbon nanotubes (SWNTs) of different diameter by Hersam’s group [71]. By wrapping DNA, they increased the hydration shell thickness and enlarged the density difference of SWNTs. It is worth noting that, the isopycnic separation exhibits high separating capability, since the density difference of different fractions of SWNTs is extremely small (1.11–1.17 g/cm3) even after the modification of DNA according to the calculation (Fig. 4).

Fig. 4 Redistribution of iodixanol and SWNTs during ultracentrifugation. (a) Profile of the density gradient before (dotted line) and after (solid line) centrifugation. During centrifugation, the iodixanol redistributed. (b-f) Sedimentation of SWNTs in a density gradient before and after 3.5, 7, 8.75, and 10.5 h of ultracentrifugation. Reprinted with permission from Ref. [71], Copyright (2005) American Chemical Society.

Based on the finding of the isopycnic mode applied on the separation of nanotubes, Hersam’s group [59] further used bile salts (e.g., sodium cholate), the planar anionic surfactants, to encapsulate and modify the SWNTs and increase their hydration shell thickness, while keeping their electronic structure intact. With the high separating capability of isopycnic separation, mixture of SWNTs, black in color due to absorbance over almost all bands in visible range, can be isolated into different chirality or diameter (e.g., with the purity of ~97% or 0.02 nm-diameter deviation), and consequently the different colors corresponding to different chirality would be seen (Fig. 5a), because the different diameter or/and chirality nanotubes exhibited

different colors. For example, the chiralities of SWNTs with diameters of 7.6, 8.3 and 9.8/10.3 Å were found to be (6,5), (7,5) and (9,5)/(8,7), corresponding to the color bands of pink, green and light brown, respectively [59]. Also, they used isopycnic separation to separate double-wall nanotubes (DWNTs) from mixtures of single- and multi-wall nanotubes through differences in their density, which could be used in transparent conductors. They added approximately 70% DWNTs into SC aqueous solution. Then the CNTs were efficiently sorted from polydisperse mixtures to monodisperse SWNTs with different diameters and DWNTs (Fig. 5b) [72], and bandgap or electronic type difference [53, 73, 74]. Optical absorption measurements of sorted DWNTs revealed that outer-wall purities of semiconducting and metallic samples could reach 96% and 98% [75]. In 2012, they presented a dual-iteration DGUC sorting strategy for isolating monodisperse metallic SWCNTs of selectable diameter via the electric arc discharge method [76]. Similarly, Maruyama’s group [77] clarified that the bile salt sodium deoxycholate (DOC) and the anionic salt sodium dodecyl sulfate (SDS) play an important role in changing the net density of carbon nanotubes and optimizing DGUC effect. Doorn group [78] also realized the effective isopycnic separation by tuning the interfacial dynamics through NaCl addition and temperature change without any additional cosurfactant.

Fig. 5 The separation of SWNTs with different diameters with DGUC. (a) Sorting of SWNTs by diameter, bandgap and electronic type using DGUC. (a1) Schematic of the surfactant encapsulation and sorting. Visually, the separation is made evident by the formation of colored bands (a2) isolated SWNTs sorted by diameter and bandgap as a resultant bundle type (a collective form of SWNTs) material is observed, aggregates and insoluble material sediment to lower in the gradient. The spectra (a3) of SWNTs is indicating that SWNTs of increasing diameter are more concentrated at the larger densities. Reprinted with permission from Ref. [59], Copyright (2006) Nature Publishing Group. (b) Separation of carbon nanotubes by a number of walls using density differentiation. (b1) Schematic illustration of carbon nanotube encapsulation by sodium cholate and its effect on nanotube buoyant density. (b2) Photograph of a centrifuge tube following the first-iteration DGUC separation of as-received nanotubes. (b3) Optical absorbance spectra of the bands of material taken from the centrifuge tube at the locations indicated. Reprinted with permission from Ref. [72], Copyright (2009) Macmillan Publishers Limited.

In 2009, Hersam’s group [64] extended this method to the layer-dependent separation of graphene. Similar to previous work, they encapsulated sodium cholate on the surface of graphite to enlarge the hydration shell thickness (h) and thereby tailor the net density (ρp) difference of graphene with various layers in aqueous solution (Fig. 6a). The characterization of atomic force microscopy and Raman spectra (Fig. 6b, c) [64] further verified the effective separation.

Fig. 6 The separation of GOs with different layers of DGUC in aqueous media. (a) Photograph of a centrifuge tube following the first iteration of density gradient ultracentrifugation. (b) Representative AFM images of graphene deposited using fractions f4 and f16 onto SiO2. In the below Height profile of regions marked in panels f4 and f16 demonstrating the different thicknesses of graphene flakes obtained from different DGUC fractions. (c) Representative Raman spectra of sorted graphene flakes from fractions f4 (blue), f10 (orange), f16 (red), f22 (purple), and f28 (green) on SiO2 with G band intensity normalized to unity. Reprinted with permission from Ref. [64], Copyright (2009) American Chemical Society.

Additionally, alternating gradient media that can also be used as targeting objects are changed. For instance, Kang et al. [79] added cesium chloride (CsCl) to iodixanol, which could enlarge the density range, to sort the relatively high-density species (rhenium disulfide). In another case, by modulating the pH during DGUC, metallic or semiconducting SWCNTs can be enriched using Pluronic copolymer as the surfactant [80]. 4.2 Rate zonal separation in aqueous media As discussed above, isopycnic separation can be used for the separation of carbon nanotubes, graphene and so on. While the isopycnic separation is usually timeconsuming and the density gradient of the aqueous solution is usually less than 1.4 g/cm3, which is far less than the net density of relative heavy nanostructures, such as noble metal nanocrystals [62], metal oxide [81], and core-shell quantum dots [82]. For such “heavy” samples, rate zonal separation method is an efficient way and has been widely used for the investigation of nanostructures. Interestingly, the rate zonal separation also started from sorting carbon nanotubes (NTs), but with much lower density gradient media to ensure the nanotubes could pass through. Sun et al. [83, 84] used aqueous solutions of iodixanol as gradient media, with the density range from 5% to 15%, to separate SWNTs (net density equals to ~20%). As can be seen from Fig. 7a, the SWNTs were fully expanded in the tube after 2 h centrifugation. The average length of obtained fractions increased from 7.9 to 61.8 nm, and f20 (the 20th fraction, as marked in Fig. 7b) contained a mixture of bundles (an aligned collection form of SWNTs) as collected in the bottom gradient.The SWNTs with different length showed different optical absorption and emission [85]. It is worth noting that, the length of f7, (7.9 ± 4.8) nm is the shortest SWNTs which cannot be prepared by mere synthesis, indicating the superior capability of rate zonal separation. In addition, they even can further narrow the length distribution of resulting NTs by applying a secondary separation. They also extended the method to separate graphene oxide nanosheets and chemically reduced graphene oxide nanosheets (Fig. 7c) [86, 87]. The obtained fractions with different size presented

specific properties, which is important for the various applications. As revealed by AFM (Fig. 7d), the size of the GO sheets distributed from 40 nm (f5) to 450 nm (f30). Correspondingly, the absorbance of GO sheets increased in the visible range with the size increasing (Fig. 7e).

Fig. 7 The separation of SWNTs and GO with different sizes with DGUC in aqueous media. (a) Schematic illustration of step density gradient. (b) AFM images of SWNTs of typical fractions as labeled. Reprinted with permission from Ref. [83], Copyright (2012) American Chemical Society. (c) Digital camera images of the ultracentrifuge tubes : before separation; GO separated in a 20%–66% gradient after separation at 50K rpm for 15 min. (d) Tapping-mode AFM images (2 μm×2 μm, scale bar: 500 nm) of GO fractions separated in a 20%–66% gradient for 15 min. (e) UV-vis absorption spectra of GO in different fractions. Reprinted with permission from Ref. [86], Copyright (2010) American Chemical Society

Besides carbon nanostructures, Sun et al. [62] also separated FeCo@C nanoparticles. They encapsulated the nanoparticles with polyethylene glycol (PEG), and enlarged the hydration shell thickness, namely, enlarged the density difference of nanoparticles. With the iodixanol/water solutions (20%+30%+40%+60%) as density gradient media and rate zonal separation method, the ~4 nm FeCo@C nanocrystalline coated with PEG were efficiently separated from 1.5 to 5.6 nm with high monodispersity (Fig. 8a and b). And then they used the polydisperse Au nanoparticles with three diameters, 5, 10 and 20 nm, respectively, to verify the feasibility and high efficiency of rate zonal separation in aqueous media (Fig. 8c and d) with a theoretical model assay (Fig. 8e).

Fig. 8 The separation of nanoparticles in aqueous media. (a) Optical and TEM images showing the separation of 4 nm FeCo@C nanoparticles. Digital camera images of ultracentrifuge tubes taken at 30 min intervals. (b) TEM images of different fractions labeled in (a). Scale bars: 50 nm. (c) DGR separation of Au NPs and recovery of mixed Au NPs. Digital camera images of ultracentrifuge vessels containing Au NPs. From left to right: 5, 10, 20 nm, and mixed. (d1-d3) TEM images of standard 5, 10, and 20 nm Au NPs; (d4-d6) TEM of images of fractions f4, f10, and f29 following separation of a mixed Au NP colloidal suspension, demonstrating unmixing of the original Au NPs. Scale bars: 50 nm. (e) The separation of different-sized colloids in a multilayer step density gradient. d = sedimentation distance. Reprinted with permission from Ref. [62], Copyright (2009) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differently shaped particles with surfactants possess different net densities in a density gradient, which can also be sorted by rate zonal separation. Akbulut et al. [88] separated the gold nanoparticles from the nanorods (Fig. 9). Small nanorods located at the top, small nanospheres in the middle, and bigger particles/large rods located at the bottom. Under optimized condition, the content of nanorods got enriched from 48% to 99% [88]. Fig. 9 The shape separation in aqueous media. (a) The evolution of the penetration of nanoparticles into an aqueous three-phase system composed of Brij 35 (8.7%, w:v), PEOZ (10%, w:v), and Ficoll (11.7%, w:v) with time during centrifugation at 16,000 g. (b) TEM images of suspension of nanoparticles (suspension of NP) and samples collected from the layers. Reprinted with permission from Ref. [88], Copyright (2012) American Chemical Society.

The rate zonal centrifugation method also shows the capability of separating nanoclusters. Clustering of three to five primary smaller magnetic nanoparticles (2 –5 nm) at bottom layer has been observed, which sedimented at the same speed with big nanoparticles (the nanoparticles with diameter ~7 nm) [62].This implied that such a procedure could also separate clusters from individual NPs, and even separate clusters depending on primary particle numbers. Chen et al. [89, 90] further developed the nanoseparation method by demonstrating this point. Gold nanoparticles with strictly the same size were firstly encapsulated with amphiphilic diblock copolymers (PSPAA) to form core-shell nanoassemblies. Based on the rate zonal separation, AuNP dimers and trimers were separated with high purity. Since structural intact is critical for the construction of any nanoassembly, the approach offers a facile separation method without additional inconvenience. Their subsequent research revealed that the structural uniformity of the “hot spots” therein reduced the ambiguities in calculating and interpreting the respective SERS enhancement factors, and the relative intensity ratios of the nanoclusters (I2NP=16I1NP and 87I3NP) involve few assumptions and are thus more reliable [90].

Fig. 10 The cluster separation in aqueous media. (a) A typical setup of differential centrifugation, where 62% and 11% aq. CsCl and then AuNP n@PSPAA in water were layered from bottom to top. (b) The result of (a) after 20 min centrifugation. (c) Separation result of a pre-enriched trimer sample. (a1, b2, and c3) TEM images of the respective fractions indicated in (a)-(c) (see large-area views in the Supporting Information online); the histograms are shown in the insets. Scale bars: 100 nm. Reprinted with permission from Ref. [89], Copyright (2009) American Chemical Society. (d) SERS spectra of the samples enriched with (I) monomers, (II) dimers, and (III) trimers of Au@Ag NPs (d=20 nm; excitation: 785 nm at 290 mW; insets: the histograms of these samples). The schematics in the lower panel show the SERS

intensity ratio of the nanoclusters. Reprinted with permission from Ref. [90], Copyright (2010) American Chemical Society.

4.3 Rate zonal separation in organic media Though powerful, aqueous separation of nanoparticles has several limitations: (1) It is only suitable for the aqueous soluble nanoparticles, while a lot of nanoparticles were synthesized in the organic phase and got solubilized therein (such as Au, CdSe, and Si nanocrystals), and phase transfer might cause serious aggregation. (2) A large mass ratio of salts or solutes is added inside to make the gradient, which significantly complicated the consequent purification procedure to obtain separated nanoparticles. Such separation requirement promotes the emergence of organic phase separation. Therein, the gradient media are an organic phase (polar or non-polar) to avoid the aggregation of dispersed colloidal nanoparticles and keep them isolated. Besides, after sampling the product out, the organic media can be vaporized to get “pure” sample. Since unbounded surfactants or soluble by-products could be isolated from the colloidal products, the rate zonal separation method can also be used for purification of nanocrystals in the organic phase. Sun’s group [91] first introduced the organic density gradient into rate zonal ultracentrifugation to sort the organic phase synthesized Au nanoparticles samples with wide size distribution. By using cyclohexane and carbon tetrachloride formulated as the organic density gradient, they separated the Au colloidal nanoparticles synthesized in oleylamine. The average sizes in fractions after separation were 4.8, 7.2, 8.0, 9.3 and 10.9 nm, with errors less than 1.5 nm (Fig. 11). Besides, with the same organic density gradient, Au nanowires were also purified from nanoparticles [91]. Further, Au nanowires can be encapsulated by copolymers (e.g., polystyrene-block-poly (acrylic acid)) to form mechanical energy stored “nanospring” on the water/oil interface [92].

Fig. 11 Digital images of ultracentrifuge vessels containing Au nanoparticles before (left vessel) and after (right vessel) separation at 25,000 r/min for 12 min and corresponding TEM images of typical fractions. The graph in the bottom right corner shows a comparison of the size distribution difference before (red columns in the upper section) and after (colored columns in the lower section) centrifugation separation. Each size histogram was measured from at least 200 particles. Reprinted with permission from Ref. [91], Copyright (2010) American Chemical Society.

The rate zonal centrifugation method cannot only separate nanomaterials with different diameters, but also with different shapes and sizes. The nanomaterials with the same component usually have the same density, but their different shapes lead to different viscosities in the solution system even with the same buoyancy (or particle volume), which can be the reason for the separation of CdS nanorods [63], as shown

in Fig. S2 (online). As a typical organic solvent, ethylene glycol is miscible with water and other organic solvents, such as ethanol and glycerol, and thus is widely used in density gradient centrifugation for both aqueous and organic systems. By mixing ethylene glycol and ethanol, Amendola’s group [65] sorted both Au and Ag nanoparticles by rate zonal separation, achieving the metal nanoparticles with very low polydispersivity. Furthermore, based on the rigorous mathematical derivation, their results suggested that rate zonal separation is one of most accurate size sorting techniques to achieve separation of metallic nanoparticles with rather limited deviation. 4.4 Isopycnic separation in organic media Similar to isopycnic separation in aqueous media, the isopycnic separation in organic media can also be achieved by replacing aqueous gradient with an organic gradient. For example, the single-walled carbon nanotubes with different chirality present different net density due to various interactions with surfactants or solvents [93]. In a typical procedure, the SWNTs with different chiralities can be dispersed in toluene with the help of fluorine-based polymers. The mixture was isopycnicly sorted in the organic solvents with 2,4,6-tribromotoluene as the density gradient additive and centrifuged with the density range of 1.2 to 1.5 g/cm3 and 50,000 r/min at 15 oC. Different nanotubes with the chirality of (7,5), (7,6), (10,5), and (9,7) were obtained with the purity of ∼90%, as estimated from the optical absorbance and photoluminescence spectra [59]. The method can also be extended to silicon nanocrystals [5]. The silicon nanoparticles (ncSi) obtained through the grinding and HCl etchings were polydispersed. As well dispersed in a given solvent, differently sized ncSi were endowed with various net density (Eq. (2)), providing the possibility to separate isopycnicly [94-96]. Ozin group [97] separated polydisperse alkyl-capped ncSi using organic density gradient ultracentrifugation. The self-generating density gradient of 40 wt.% 2,4,6-tribromotoluene in chlorobenzene was used due to the good suspension and density matching. The polydispersity index values of isolated fractions were found to be as long as 1.04–1.06 based on photoluminescence spectroscopy and scanning transmission electron microscopy characterization, as shown in Figs. 12 and S3 (online).

Fig. 12 The separation of silicon nanocrystals (ncSi) in organic media. (a) Decylcapped ncSi: schematic of decyl-capped ncSi, the image of a colloidal suspension of decyl-capped ncSi in hexanes under ambient light (left) and photoexcitation (right) and UV-vis absorption (orange), PL (red), and excitation (black) spectra of ncSi. (b) The schematic of ultracentrifugation for sorting ncSi. Reprinted with permission from Ref. [97], Copyright (2011) American Chemical Society.

4.5 Viscosity dependent separation Based on Eq. (10), we can see that the rate zonal separation mainly relies on the sedimentation velocity which can be determined by using an external diameter of particles (V−(r+t)2), the isopycnic separation mainly relies on the density difference of particles (V=0 when ρp=ρm). While in some conditions, we can change the viscosity of gradient media by introducing polymer without influencing density. Sun’s group [91] used the cyclohexane/carbon tetrachloride density gradient to separate the cadmium selenide quantum dots, and they studied the viscosity’s influence on the separation effect by adding the polystyrene (PS) in the density gradient medium. As the introduction of PS into the organic gradient layers, it can significantly increase their viscosity; it should slow down the sedimentation of nanoparticles. As expected, PScontaining gradient (vessel II) showed a limited separation compared to the PS-free gradient (vessel I), and only by applying longer centrifugation time, the separation can be completed (vessel III), and thus a finer separation can be achieved (Fig. 13) [91]. This work demonstrated the possibility to separate tailoring media viscosity.

Fig. 13 The separation of CdSe NPs in different viscosity systems. (a) Digital camera images of ultracentrifuge vessels containing CdSe NPs: (vessel I) PS-free gradient, 60 min centrifugation at 50,000 r/min; (vessel II) PS-containing gradient, 60 min centrifugation at 50,000 r/min; and (vessel III) PS-containing gradient, 110 min centrifugation at 50,000 r/min. All images were recorded under UV irradiation at 365 nm. (b) Digital image of composite strips converted from PS-containing fractions of CdSe NPs with different sizes (size: 105 mm × 20 mm) (c) Fluorescence spectra of typical fractions obtained from vessel I, PS-free gradient. (d) Fluorescence spectra of fractions obtained from vessel III, PS-containing gradient. Reprinted with permission from Ref. [91], Copyright (2010) American Chemical Society.

Further, Qiu and Mao [98]verified that the viscosity gradient is a novel and effective method for separating nanoparticles. Polyvinylpyrrolidone (PVP) is a highly aqueous soluble polymer with a relative low cost to make the viscosity gradient. There was trivial density change of water (1.064 g/cm3 at 30 wt.% PVP), while the viscosity changed dramatically when the PVP concentration varied [99]. They successfully built up the 10 wt.%–30 wt.% aqueous PVP solutions to separate a small volume of concentrated Au nanoparticles solution. As shown in Fig. 14, after centrifuging at 3,400 g for 2.5 h, five fractions can be separated clearly in the gradient media. TEM images (Fig. 14 a–e) showed that the uniformly distributed Au nanoparticles were 15, 18, 21, 27, and 31 nm for each fraction. Larger Au nanoparticles can be well separated in a relatively high viscosity gradient (such as 20 wt.%–40 wt.% aqueous PVP solutions). They also employed the PVP viscosity gradient to separate and purify the polydisperse iron oxide nanoparticle clusters which were synthesized by using selective phase evaporation in a CTAB-based on oil-in-

water emulsion system [98]. Although, if the particles were smaller than 30 nm it can be obtained by differential centrifugation [100], the rest part were distributed from 30 to 150 nm.

Fig. 14 Separation of 15, 18, 21, 27, and 31 nm five differently sized Au NPs in a PVP viscosity gradient. Photographs were taken at 0.5 h intervals. (a-e) TEM images, corresponding to fractions from top to bottom, show NPs size in each fraction. Scale bar in (a-e) is applicable to all images. Reprinted with permission from Ref. [98], Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

4.6 Other dependent separation As demonstrated by above examples, the optimization strategies of DGUC can be commonly divided into two sections. On one hand, modification/encapsulation of the target nanoparticles with ligands/surfactants/polymers can be used to enlarge the net density difference for the separation. On the other hand, introducing some highviscosity solvents (e.g., ethylene glycol, EG in brief) or polymer additives (e.g., PVP and PS) can increase the media viscosity to promote the separation precision. It is common that the density and viscosity of gradient gets increased simultaneously. For instance, by mixing EG and ethanol, both density and viscosity gradientare naturally formed. Bonaccorso et al. [65]separated laser ablation-synthesized Au and Ag NPs with it. Similarly, CdSe nanoparticles were also sorted in cyclohexane/CCl4 gradient with PS as the additive [91]. Apart from these, some other properties-dependent strategies, such as polarity and hydrophilicity have also been adopted for the separation by density gradient ultracentrifugation. For instance, Deng et al. [101] have introduced a hydrophilicity gradient to separate non-sedimental CDs, which were pretreated by acetone to form clusters. Such clusters “de-clustered” (release primary particles) as they sedimented through media comprising gradients of ethanol and water with varied volume ratios. During the centrifugation, primary CDs with the varied sizes and carbonization degrees detached gradually from the clusters to get well dispersed in the corresponding gradient layers [101]. Their settling behavior highly depends on the varied hydrophilicity and solubility of the environmental media, which evidenced the wide feasibility of the DGUC method. 5. Extension of DGUC: “lab in a tube” Density gradient centrifugation has been established to obtain monodisperse nanoparticles with strictly uniform size and morphology, which are usually hard to be got by merely synthetic optimization. The above sections have demonstrated the

versatility and universality of such separation method, by which nearly all kinds of nanostructures can be separated, including zero, one and two-dimensional nanomaterials. Further, reaction mechanism can also be investigated based on the separated fractions. The focus of this section is the reaction mechanism analysis using density gradient centrifugation. The concept of “lab in a tube” was first put forward as a micro-total analysis system in the field of biology [102, 103]. In recent decades, in order to extend the functionality of old lab-on-a-chip system [104], lab-in-a-tube has been designed to compress an entire laboratory into a smaller architecture, in which lots of individual detection or analysis components were integrated and each could be used individually or together [102, 105]. In 2012, Sun’s group [106] extended to this concept in the field of nanoseparation. “Lab in a tube” based on nanoseparation is the integration of numerous functional gradient layers into a single centrifuge tube constituting a microsystem of several independent units, each can individually perform its specific role such as “reaction zone” or “assemble zone”. 5.1 For quick surface reaction investigation To date, the investigation of the surface reaction mechanism still critically relies on the capture of reaction intermediates [107-109]. However, the high surface area of NPs endows NPs with high reactivity, which lead to a quick reaction rate; unfortunately, such intermediates are usually hard to be obtained in a short time through traditional centrifugation methods. DGUC nanoseparation method provides the new opportunity of isolating intermediate NPs within a short period of time. By introducing a reaction zone in gradient layers, surface reaction mechanism can be investigated, since the reaction time can be finely controlled and the chemical environment can be changed very soon. As an example, to investigate the surface reaction mechanism of galvanic replacement reaction between Au and Ag, a 20% to 70% EG/H 2O gradient layers were used in centrifugation [106]. As shown in Fig. 15a, the second layer was set as reaction zone by introducing a certain amount of reactant “HAuCl4” and the first layer was used as a buffer layer to prevent a direct mixing and reaction. Furthermore, the lower four layers acted as the separation zone to separate the reacted Ag nanoplates by their sizes. Ag nanoplates prepared in the aqueous phase was placed on the top of the gradient layers. After separation, a clearly red-shifting could be observed, demonstrating the increased size of reacted Ag nanoplates from f6 to f13. However, f5 did not show the similar trend, which should be attributed to the hollow structures.

Fig. 15 Investigation on the surface reaction mechanism using “lab in a tube” method. (a) Schematic illustration of surface reaction mechanism investigation by introducing a reaction zone. (b1) TEM images of the reacted Ag nanoplates in f9. The insets are magnified images (scale bar: 20 nm). (b2) TEM image of Ag nanoplates after galvanic reaction without DGUC. (c) Schematic illustration of the structural evolution of a

triangular Ag nanoplate during the surface reaction. Reprinted with permission from Ref. [106], Copyright (2012) The Royal Society of Chemistry.

The hollow structure of Ag nanoplates in f5 was further confirmed by TEM, as shown in Fig. 15b. Ag nanoplates with smaller size have slower sedimentation rate, which could lead to a longer exposure time and thus result in their hollow structures. On the contrary, bigger Ag nanoplates have a shorter exposure time, which results in a short-time reaction. As estimated, the exposure time of f9 in reaction zone was only (30 ± 17) s, which is much shorter than that which can be achieved by the traditional reaction and centrifugal process (Fig. 15b). UV-vis spectroscopy of the no separation sample showed a broad in-plane dipole SPR band, suggesting the wide size distribution of reacted Ag nanoplates. To get a deeper insight into the surface reaction mechanism, the Ag nanoplates in f9 were characterized by HRTEM and EDS (Fig. 15c). The Au/Ag atomic ratio of the edge regions was measured to be 0.231, much higher than other regions, indicating the edge side should be the favored site for such surface reaction. Besides, the Au/Ag atomic ratio of thick and thin part of the basal plane was 0.064 and 0, respectively. This means, the thin part presents the Ag dissolution zone and the thick part should be responsible for the Au deposition. On the basis of the results above, the structural evolution of triangular Ag nanoplates during the surface reaction can be divided into two stages, as shown in Fig. 15c. At the initial stage, the reaction starts at the edge side and at the same time on the basal plane surface. With the reaction time increasing, Ag dissolution and Au deposition jointly lead to the formation of the hollow structure when the reaction comes to its end at stage II. 5.2 To understand the reaction environment/reagent roles The crystallization behaviors of inorganic nanostructures are often different even in one-pot synthesis since the microenvironment of their nucleation and growth cannot be exactly the same. Synthetic optimization is traditionally based on repeated control or crossing experiments. In view of the fact that nanoparticles with different size, morphology or phase can be separated through DGUC by analyzing the chemical composition or crystal structure of separated fractions, key pieces of information can be obtained and thus guide synthetic optimization [63]. Cadmium sulfide (CdS) semiconducting nanorods have a Band gap of 2.4 eV with photoluminescence at 514 nm. We have separated CdS nanorods by DGUC and obtained CdS nanorods with precisely tailored electrical and optical properties, inspired by nanoseparation [63, 110, 111]. After centrifugation in a cyclohexane plus tetrachloromethane gradient, longer CdS nanorods could be observed at lower positions of the centrifuge tube, and shorter could be found at the top, as shown in Fig. 16a. The relationship between the length of CdS nanorods and their photoluminescence properties turned out after separation, which inspired us to selectively enrich specific samples by synthesis optimization. Oxygen was revealed

by a dominating role in tailoring photoluminescence of the final product. Further research showed that the crystal structure of CdS is a hexagonal phase at the presence of sufficient oxygen, while cubic phase formed with insufficient oxygen (Fig. 16b, c) The CdS nanorods in N2, air, and O2 atmosphere were synthesized to support this conclusion. As expected, N-CdS synthesized in N2 contained the shortest nanorods with photoluminescence dominated by short wavelength band-edge emission whilst O-CdS NRs were the longest and long wavelength surface-trap emission predominated.

Fig. 16 Exploring the impact of the reaction environment. (a) Digital camera image of ultracentrifuge vessels containing CdS nanorods after centrifugation, corresponding TEM images and photoluminescence spectra of typical fractions (inset: the length of different fractions). Reprinted with permission from Ref. [110], Copyright (2010) Tsinghua University Press and Springer-Verlag Berlin Heidelberg. (b) Digital camera images of the ultracentrifuge tubes after separation at 30,000 r/min, HRTEM images and electron diffraction patterns of f14 and f37 (insets: fast Fourier transform (FFT) patterns). Reprinted with permission from Ref. [63], Copyright (2011) American Chemical Society. (c) Schematic illustration of an effective oxygen on the formation of CdS nanorods. Reprinted with permission from Ref. [111], Copyright (2012) American Chemical Society.

Chang et al.[112] adopted the density gradient centrifugation to separate the Mg/Al LDH nanosheets. Through the study of fractions with varied lateral sizes, it could be concluded that the Mg/Al proportion of the products varied with size: larger nanosheets have a higher Mg/Al molar ratio. Through the analysis of the structure, size and composition of the products by the DGUC method, the growth mechanism of the 2D materials turned clearer. The crystal growth speeds of LDH nanosheets varied according to local concentration; when these sheets formed at Mg-rich locations they grew fast, while others formed at Mg-lean locations grew slowly, which led to the presence of LDH nanosheets with different size of particles and compositions in the same system. Sun’s group [113] also studied the phase transition of Yb3+ and Er3+ co-doped NaYF4 nanocrystals (NaYF4:Yb3+/Er3+NCs) using density gradient ultracentrifugation separation. The NaYF4:Yb3+/Er3+NCs with mixed cubic and hexagonal phases were synthesized in an oleic acid-water-ethanol system via the hydrothermal process. After separation, the fraction f1 (small cubes, ~28 nm) was orange colored under excitation at 980 nm, and the middle fraction f6 (big cubes~44 nm) turned red whilst fraction f18 (nanorods ~1.2 μm) at the bottom exhibited green emissions, as shown in Fig. S4 (online). Elemental analysis revealed that small cubes (f1) with a higher Y content were formed at the initial stage, the subsequent phase transition (stage II) led to the formation of thermally stable β-phase nanorods, which were rich in Yb. After the

dissolution-crystallization equilibrium (stage III), the Y:Yb:Er atomic ratio turned the same as the feeding ratio, and the nanorods became bigger in size. 5.3 How “assembly/aggregation” initiate Controlled assembly of NPs is critical for the investigation of their collective properties, which is of great importance in guiding the fabrication of elaborate nanodevices [90, 114]. However, random Brownian motion remained the only way to achieve the symmetric assembly of NPs and such uncontrollable method has greatly limited the application of the assembled structures. Since centrifugal field can be applied to overcome the Brownian motion effect of NPs, DGUC separation can be designed to make colloidal hetero-assembly by introducing an “assembly zone” in the density gradient layers [115]. During the centrifugal process, the directional motion of bigger NPs should be faster than that of NPs with smaller size and thus asymmetric hetero-assemblies can be fabricated by a “crash reaction” as schematically shown in Fig. S5a (online). Big Au NPs, ~60 nm in diameter and with a positively charged surface, were placed on the top of the density gradient layers. Meanwhile, ~20 nm Au NPs with a negatively charged surface were set at a lower layer, with a buffer layer inserted to avoid a spontaneous assembly. When a large centrifugal force was applied, big Au NPs can cross through the buffer layer and crash on the small ones with opposite charge (Fig. S5b online) [116]. Fig. S5d (online) shows a typical result of asymmetric assembly in the density gradient. After the “crash reaction”, the UV-vis spectra showed a slight red-shift from f18 to f22, demonstrating the successful assembly of Au NPs. Besides, TEM images in Fig. S5e-f (online) further confirmed the asymmetric assembly.

Fig. 17 Understanding the assembling of Au NPs. (a) Illustration of the assembly and separation of Au NPs in a density gradient centrifugation system with a PAA layer. (b) Digital camera images of centrifugation tube before and after centrifugation. (c) TEM images of four typical fractions: f1, f3, f5, and f7 (scale bars: 100 nm), HRTEM image of the nanostructure (scale bar: 5 nm), and UV-vis spectra of AuNPs and the four fractions. Reprinted with permission from Ref. [115], Copyright (2015) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zero-dimensional nanoparticles can self-assemble into 1D nanostructures by some interaction force between nanoparticles [117]. The NaCl solution can change the electrostatic interactions between Au nanoparticles. Qi et al. [115] introduced a thin NaCl solution layer on the way of Au nanoparticle sedimentation (between original Au colloidal solution and gradient) to achieve the controllable assembly of Au nanoparticles into one-dimensional nanostructure and consequent separation (Fig. 17). In order to encapsulate or functionalize the Au nanostructures, they introduced a relevant charged polymer layer following NaCl solution layer. They got a different

length self-assembly Au nanostructure, which showed distinguishable UV/Vis absorption or gives tunable fluorescence properties. This method provided a very facile and controllable way to obtain separated assembled colloidal nanoparticles. 5.4 Ultraconcentrate nanoparticles Many reactions involved the switching between the aqueous and oil phase systems and quasi-homogeneous catalysis need recollection of colloidal nanoparticles [118]. We introduced oil-water interface in the centrifuge tube to separate and collect nanostructures. Kuang et al. [119] had a detailed study in this aspect. The behavior of nanoparticles at water/oil interface is found to be very different from single phase, and “super-concentrated droplets” could form (Fig. 18a). There are basically two kinds of water/oil interface DGUC systems, oil-aqueous-oil and aqueous-oil-aqueous. Using those systems, the solvent switching of nanoparticles can be achieved, which made the recollection of quasi-homogeneous nanocatalyst possible. During this switching, it is noticed that the concentration of purified products can be achieved by changing the volume ratio between the top and bottom layer (Fig. 18b). We thus minimized the volume of the bottom layer to zero, and finally got the ultraconcentrated droplets (Fig. 18c). As estimated, the volume of the droplet is even less than 0.3 μL, which means ~10,000 times of concentration increase, or, >60% volume percentage.

Fig. 18 Ultraconcentrate nanoparticles using DGUC. (a) Schematic of solvent switching and concentration control of the droplet sedimentation. (b) Digital camera images aqueous Au solutions before and after solvent switching . (c) Digital camera images of aqueous Au solutions before and after concentration, linear fitting of Vn vs. Coriginal/Cn to calculate the concentration increase and total volume brought down. Reprinted with permission from Ref. [119], Copyright (2014) Tsinghua University Press and Springer-Verlag Berlin Heidelberg. Water/oil interfaces centrifugation method not only have high purification efficiency but also have a selectivity of the sample size and morphology. The difference in upper and bottom layer after separation which contains both 20 and 50 nm Au nanoparticles in the upper layer. UV-Vis spectra indicate that the upper layer was 20 nm Au nanoparticles and only 50 nm Au nanoparticles pass through the interface under low-speed centrifugation. Ultra-concentration method of colloidal NPs through water/oil interfaces is not only suitable for aqueous phase but also an organic phase. Depending on density gradient and interfacial blocking, different morphologies nanoparticle can be concentrated and purified without aggregation. Furthermore, as mentioned above, larger than 60%volume of ultra-concentration colloid was occupied by Au NPs, such one-step ultra-concentration method has selectivity for the different sizes and

morphologies, meanwhile could remove the 99.99% impurity one time only. Such high-efficient way is better than any other separation method. 6. Concluding remarks and perspective Monodispersity of nanomaterials is the basis for studying the size effect and surface effect. DGUC is a high-efficiency liquid phase separation and purification technology that has been demonstrated by a powerful tool to sort various nanomaterials. The colloidal nanostructures are kept in the solution phase during the whole process, which can effectively avoid the aggregation and property change. The separation can be conducted in aqueous or organic, polar or non-polar phase, and be used for the purification of metals, metal oxides, metal sulfides, carbon materials and semiconductor materials, etc. Colloidal nanostructures can be separated according to size, net density, morphology or other characteristics without sample loss. Furthermore, by separation parameter optimization, the separation efficiency can be further improved. In this review, the explorations of the DGUC method fully show it as a promising separation and purification method for the nanomaterials. Moreover, by introducing the “reaction zone” in the centrifuge tube for different density gradient systems, one can achieve “lab in a tube” method, which can capture the intermediate species and pave an effective way to study the reaction/growth mechanisms, aggregation behaviors, and component difference and so on. Thus, DGUC technology can be applied as a powerful “post-synthesis” tool to achieve strictly monodisperse nanostructures, and also be capable for providing the inspiration for the growth/reaction mechanism, which should be a new direction in the field of nanotechnology to promote the further investigations and applications of nanomaterials. In spite of the improvements, there are still some challenges. For example, the facet-dependent crystals and the exact magic number nanoclusters separation are still hard. And, the large-scale application with low energy and timecost still requires further efforts. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC), the National Key Research and Development Project of China (2016YFF0204402), the Program for Changjiang Scholars and Innovative Research Team in the University (IRT1205), the Fundamental Research Funds for the Central Universities, the Long-Term Subsidy Mechanism from the Ministry of Finance and the Ministry of Education of PRC. References

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Kuang Y, Song S, Liu X, et al. Solvent switching and purification of colloidal nanoparticles through

water/oil interfaces within a density gradient. Nano Res 2014; 7: 1670-1679

Pengsong Li is now pursuing his Ph.D. degree under the supervision of Porf. Xiaoming Sun at the State Key Laboratory of Chemical Resource Engineering, College of Energy, Beijing University of Chemical Technology. His current research interests include synthesis and separation inorganic nanostructures in their application of energy conversion and storage.

Liang Luo achieved his B.S. at Beijing Normal University at 2005, and his Ph.D. at Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences at 2010. He joined Beijing University of Chemical Technology from 2010. His current research interest mainly focuses on the synthesis and separation of nanomaterials, and the in situ characterization of electrocatalysis.

Xiaoming Sun achieved his B.S. and Ph.D. at Tsinghua University at 2000 and 2005, respectively. He worked in Stanford as a postdoc from 2005. He joined Beijing University of Chemical Technology from 2008 and was supported by Foundation of Outstanding Young Scholar from National Natural Science Foundation of China in 2011. His current research interest mainly focuses on the synthesis and separation of nanomaterials, to improve the energy related electrocatalysis process by tailoring the compositions, surface wettability, and micro-/nano-structures.

Density gradient ultracentrifugation (DGUC) is an effective separation and purification technique of nanoparticles. Introducing a reaction zone or an assembly zone in the gradient can find the surface reaction and assembly mechanism of NPs since the reaction time can be precisely controlled and the chemical environment change can be extremely fast. The ultraconcentration of NPs can also be achieved by introducing the water/oil interfaces into the separation chamber. In this review, we have emphasized the conceptual advances in classification, mechanism of DGUC and synthesis-structure-property relationships of NPs to provide the significant clue for the further synthesis optimization.