*3.1.2 Device mounting in beamline/SAXS instrument*

It should be expected that the device will be required to be perpendicular to the beam. Further consideration should also be given to the orientation of the channels, with respect to the beam dimensions. Generally, it is optimal to orient the channels so that as much of the beam is going through the channel as possible, and as little as possible is hitting the device body. This minimises background, and optimises the signal that can be achieved. In the best case scenario, the beamline will have the capability to generate micro beams of a few micron in any dimension. This allows for optimal exposure for the sample, and greatly increased time resolution in timeresolved samples.

It is best if the device has a chip-holder to mount the device in, which in most cases is specific to the setup and design. This holder must allow for any necessary connections of inlet and outlet tubing while holding the microfluidic device steady and without tension on any connections to pumps or vials. Ideally, this holder would be placed on a motor-controlled, adjustable stage to facilitate precise alignment in the X-ray beam and movement of the device to scan along outlet channels for different points in time of reaction kinetics.

In many cases, beamlines and lab instruments will maintain a vacuum along the complete X-ray flight path, and may include a vacuum sample environment. As Xrays interact with all matter, it is a requirement that there not be air in the majority of the SAXS instrument. Vacuum sample environments take this further by removing all air in the system to reduce and minimise background scattering. If a vacuum sample environment is in use, the microfluidic device must be designed to withstand the vacuum levels, and to minimise outgassing and other deleterious effects.

#### **3.2 Nanoparticle nucleation and growth**

A fundamental principle in nature and technology is self-assembly – the formation of ordered structures of components of a system out of chaotic arrangements without external forces. These processes can be induced by a multitude of parameters, e.g. change of solvent, pH, temperature, pressure or by introduction of additional reactants. SAXS, being sensitive to length scales of 1–100 nm is an ideal technique for studying nanoparticle size and structure from nucleation to the final particle. *In situ* SAXS measurements of nanoparticle synthesis is typically used to monitor the kinetics of this process [7], and increasingly incorporates microfluidics.

**Metal nanocrystals.** Metal nanoparticle syntheses is particularly amenable to SAXS analysis, as their high electron density contrast allows measurements in dilute suspensions even at the very early stages of particle nucleation. This has been employed for investigating silver (Ag) and gold (Au) nanoparticle formation and structure [28–31]. The first steps towards microfluidic setups were stopped flow measurements, for example the kinetics of gold nanoparticle formation, and the

concurrent evolution of the optical properties of the particle at room and high temperature was very successfully investigated at millisecond resolution with this method by Abécassis et al. [32, 33] and Chen et al. [26]. Further development by Polte et al. provided *in situ* studies on the nucleation and growth of Au and Ag nanoparticles in stopped and continuous flow microfluidic devices [34, 35] Free liquid jets coupled to microfluidic mixers have aided in reducing background and improving signal to noise of SAXS measurements [36].

Amphiphilic diblock copolymers show fast self-assembly processes at a timescale

Apart from surfactants, polymers and polymer coated particles, other materials

benzene tricarboxxamide) to nanofibrils of several hundred nanometres length by CLSM and SAXS. The measurement of the self-assembly process utilised 3D hydrodynamic focusing microfluidic devices (**Figure 3D-F**). Even bigger structures could be followed in the case of collagen and collagen derived fibres by pH-induced selfassembly. Here microfluidic chips provide an excellent platform for wet-spinning processes, shown by Haynl et al. [44] and Hofmann et al. [45], while SAXS can provide important information about the internal structure of the fibres during formation [46]. Furthermore, the alignment of macromolecular structures, such as worm-like micelles, patchy polymers and nanoplatelets can be investigated in (tapering) channels *on chip* [47] as well as in free jets (**Figure 3K**-**M**) [48].

*Schematics and images of microfluidic devices used for time-resolved nanoparticle nucleation and growth and macromolecular self-assembly. (A) T-shaped, single layer (B) hydrodynamic focusing microfluidic device, made from Kapton (C)* [40]. *(D) Three-dimensional (multilayer) hybrid hydrodynamic focusing device (E), made from PDMS with inserted glass capillary (F)* [24]. *(G) Double stream hydrodynamic focusing device, which can be aligned without optical access (H), made from SIFEL with a thin PDMS carrier layer and inserted glass capillary (J)* [23]. *(K) Multilayer (L) micro-jet device with hydrodynamic focused spray out of a*

,N″-tris(4-carboxyphenylene)-1,3,5-

have self-assembly properties and can be investigated with a combination of microfluidics and X-ray scattering. Seibt et al. followed the pH-induced, rapid

of seconds. These can be followed *in situ* with specially designed equipment by synchrotron-based SAXS, as shown by Stegelmeier et al. for PS-P4VP block copolymers by rapid removal of solvent [43]. An elegant way to study these fast selfassembly processes *in situ* in solution is shown by With et al. by measuring the concentration-induced lyotropic phase transition of PI-PEO polymers. Employing a simple cross-shaped multilayer Kapton microfluidic device (**Figure 3A-C**) in combination with synchrotron microfocus SAXS, time-resolved self-assembly of the used PI-PEO polymers via a spinodal microphase to micelles into FCC liquidcrystalline phases could be studied with millisecond resolution [40]. A more sophisticated channel design was used to study the self-assembly of PI-PEO block copolymers via spherical micelles into a FCC lattice (**Figure 3G**-**J**) and the solventinduced self-assembly of PEG-PLAinto spherical micelles, cylindrical micelles and

*Microfluidics for Time-Resolved Small-Angle X-Ray Scattering*

*DOI: http://dx.doi.org/10.5772/intechopen.95059*

vesicles by Fürst et al. [23].

**Figure 3.**

**27**

*nozzle, made entirely from PDMS (M)* [41].

assembly of disk-shaped hydrogelators (N,N<sup>0</sup>

When combining microfluidic setups and X-ray scattering for nanoparticle investigation, not only the reaction kinetics of nucleation and growth processes can be measured, but also agglomeration kinetics and structures. For example, Gerstner et al. combined a static microfluidic mixing device with in line absorption and SAXS measurements to study the rapid superlattice formation of alkylthiol-coated Au nanoparticles at different temperatures, which showed a differentiation between long- and short-range self-assembly effects of temperature on a time scale down to 3 seconds [37]. A further example is the time-resolved analysis of polystyrene (PS) coated Au nanoparticles by Merkens et al. in a Kapton-based 3D hydrodynamic focusing microfluidic chip, that revealed the subsecond kinetics of structural transitions involved in solvent induced collapse [38].

**Semiconductor nanocrystals.** Inorganic semiconductor nanoparticles, also called quantum dots (QDs), have received much attention due to their bright and size-tunable photoluminescence, which is commonly used as a key measurement property during synthesis [39]. During a synthesis of QDs, inorganic particles undergo a process of nucleation, growth and agglomeration, followed by dispersion into a buffer solution to quench the reaction. In order to synthesise homogeneous particles it is important to induce rapid nucleation and control the growth rate. Microfluidic devices with hydrodynamic focusing have been extremely useful in achieving this controlled synthesis process [15]. We have used 3D hydrodynamic focussing device for the synthesis of CdS nanoparticles, both studying the reaction by confocal laser scanning microscopy (CLSM) and SAXS. The CLSM measurements, using a full-PDMS device, showed the increase and shift of photoluminescence related to the nucleation and growth of CdS nanoparticles along the outlet channels. Hybrid microfluidic chips, consisting of the mixing cross section in PDMS and an inserted glass capillary as outlet channel (**Figure 3D**-**F** D-F), were developed for *in situ* SAXS measurements with low scattering background [24]. Employing a stopped flow setup, the nucleation and growth of ZnO nanoparticles was characterised at the timescale of seconds [27]. Further work elucidated the kinetics of the process at the microsecond timescale, using a free-jet device with a microfluidic T-mixer setup with a nozzle outlet to perform synchrotron SAXS measurements of the reaction in air (in the free jet). These setup enabled the investigation of QD synthesis with and without stabilising agents [42], highlighting the use of microfluidics and SAXS in the development of straightforward processes for nanoparticle synthesis.

#### **3.3 Macromolecular self-assembly**

Structural evolutions of pure and mixtures of surfactants that are often used in nanoparticle synthesis reactions, can also be investigated by a combination of microfluidic platforms and SAXS. Fürst et al. used a simple, T-shaped microfluidic chip to measure the structural assembly of tetradecyldimethylamine oxide (TDMAO) and lithium perfluorooctanoate (LPFO) in combination with synchrotron SAXS. This revealed the kinetic fusion mechanism of the cylindrical TDMAO and spherical LPFO micelles to disk-like micelles as a diffusion limited process, resulting in lamellar correlations at final stages [23].

*Microfluidics for Time-Resolved Small-Angle X-Ray Scattering DOI: http://dx.doi.org/10.5772/intechopen.95059*

concurrent evolution of the optical properties of the particle at room and high temperature was very successfully investigated at millisecond resolution with this method by Abécassis et al. [32, 33] and Chen et al. [26]. Further development by Polte et al. provided *in situ* studies on the nucleation and growth of Au and Ag nanoparticles in stopped and continuous flow microfluidic devices [34, 35] Free liquid jets coupled to microfluidic mixers have aided in reducing background and

When combining microfluidic setups and X-ray scattering for nanoparticle investigation, not only the reaction kinetics of nucleation and growth processes can be measured, but also agglomeration kinetics and structures. For example, Gerstner et al. combined a static microfluidic mixing device with in line absorption and SAXS measurements to study the rapid superlattice formation of alkylthiol-coated Au nanoparticles at different temperatures, which showed a differentiation between long- and short-range self-assembly effects of temperature on a time scale down to 3 seconds [37]. A further example is the time-resolved analysis of polystyrene (PS) coated Au nanoparticles by Merkens et al. in a Kapton-based 3D hydrodynamic focusing microfluidic chip, that revealed the subsecond kinetics of structural

**Semiconductor nanocrystals.** Inorganic semiconductor nanoparticles, also called quantum dots (QDs), have received much attention due to their bright and size-tunable photoluminescence, which is commonly used as a key measurement property during synthesis [39]. During a synthesis of QDs, inorganic particles undergo a process of nucleation, growth and agglomeration, followed by dispersion into a buffer solution to quench the reaction. In order to synthesise homogeneous particles it is important to induce rapid nucleation and control the growth rate. Microfluidic devices with hydrodynamic focusing have been extremely useful in achieving this controlled synthesis process [15]. We have used 3D hydrodynamic focussing device for the synthesis of CdS nanoparticles, both studying the reaction by confocal laser scanning microscopy (CLSM) and SAXS. The CLSM measurements, using a full-PDMS device, showed the increase and shift of photoluminescence related to the nucleation and growth of CdS nanoparticles along the outlet channels. Hybrid microfluidic chips, consisting of the mixing cross section in PDMS and an inserted glass capillary as outlet channel (**Figure 3D**-**F** D-F), were developed for *in situ* SAXS measurements with low scattering background [24]. Employing a

stopped flow setup, the nucleation and growth of ZnO nanoparticles was

of the process at the microsecond timescale, using a free-jet device with a microfluidic T-mixer setup with a nozzle outlet to perform synchrotron SAXS measurements of the reaction in air (in the free jet). These setup enabled the investigation of QD synthesis with and without stabilising agents [42], highlighting the use of microfluidics and SAXS in the development of straightforward processes

for nanoparticle synthesis.

**26**

**3.3 Macromolecular self-assembly**

resulting in lamellar correlations at final stages [23].

characterised at the timescale of seconds [27]. Further work elucidated the kinetics

Structural evolutions of pure and mixtures of surfactants that are often used in

nanoparticle synthesis reactions, can also be investigated by a combination of microfluidic platforms and SAXS. Fürst et al. used a simple, T-shaped microfluidic

chip to measure the structural assembly of tetradecyldimethylamine oxide (TDMAO) and lithium perfluorooctanoate (LPFO) in combination with synchrotron SAXS. This revealed the kinetic fusion mechanism of the cylindrical TDMAO and spherical LPFO micelles to disk-like micelles as a diffusion limited process,

improving signal to noise of SAXS measurements [36].

*Advances in Microfluidics and Nanofluids*

transitions involved in solvent induced collapse [38].

Amphiphilic diblock copolymers show fast self-assembly processes at a timescale of seconds. These can be followed *in situ* with specially designed equipment by synchrotron-based SAXS, as shown by Stegelmeier et al. for PS-P4VP block copolymers by rapid removal of solvent [43]. An elegant way to study these fast selfassembly processes *in situ* in solution is shown by With et al. by measuring the concentration-induced lyotropic phase transition of PI-PEO polymers. Employing a simple cross-shaped multilayer Kapton microfluidic device (**Figure 3A-C**) in combination with synchrotron microfocus SAXS, time-resolved self-assembly of the used PI-PEO polymers via a spinodal microphase to micelles into FCC liquidcrystalline phases could be studied with millisecond resolution [40]. A more sophisticated channel design was used to study the self-assembly of PI-PEO block copolymers via spherical micelles into a FCC lattice (**Figure 3G**-**J**) and the solventinduced self-assembly of PEG-PLAinto spherical micelles, cylindrical micelles and vesicles by Fürst et al. [23].

Apart from surfactants, polymers and polymer coated particles, other materials have self-assembly properties and can be investigated with a combination of microfluidics and X-ray scattering. Seibt et al. followed the pH-induced, rapid assembly of disk-shaped hydrogelators (N,N<sup>0</sup> ,N″-tris(4-carboxyphenylene)-1,3,5 benzene tricarboxxamide) to nanofibrils of several hundred nanometres length by CLSM and SAXS. The measurement of the self-assembly process utilised 3D hydrodynamic focusing microfluidic devices (**Figure 3D-F**). Even bigger structures could be followed in the case of collagen and collagen derived fibres by pH-induced selfassembly. Here microfluidic chips provide an excellent platform for wet-spinning processes, shown by Haynl et al. [44] and Hofmann et al. [45], while SAXS can provide important information about the internal structure of the fibres during formation [46]. Furthermore, the alignment of macromolecular structures, such as worm-like micelles, patchy polymers and nanoplatelets can be investigated in (tapering) channels *on chip* [47] as well as in free jets (**Figure 3K**-**M**) [48].

#### **Figure 3.**

*Schematics and images of microfluidic devices used for time-resolved nanoparticle nucleation and growth and macromolecular self-assembly. (A) T-shaped, single layer (B) hydrodynamic focusing microfluidic device, made from Kapton (C)* [40]. *(D) Three-dimensional (multilayer) hybrid hydrodynamic focusing device (E), made from PDMS with inserted glass capillary (F)* [24]. *(G) Double stream hydrodynamic focusing device, which can be aligned without optical access (H), made from SIFEL with a thin PDMS carrier layer and inserted glass capillary (J)* [23]. *(K) Multilayer (L) micro-jet device with hydrodynamic focused spray out of a nozzle, made entirely from PDMS (M)* [41].
