Table 1.

Commonly used and more recent methods for characterising nanocrystals.

Category	Characterisation Method	Detection Principle	Information	Data Type	Variations	Sample Requirements	Considerations	References
Solid state form	X-ray powder diffraction (XRPD)	Diffraction of x-rays from lattice planes	Polymorphic form (unique diffraction peaks), amorphous form (no peaks)	Diffractogram, qualitative and quantitative (degree of crystallinity)	Hot stage XRPD to analyse solid state form as a function of temperature	Powder, paste or slurry form, several sample presentation setups possible, amount required depends on setup	Anisotropic particle shape leads to preferred orientation effects (change in relative intensities of diffraction peaks)	[17]
							Peak broadening can occur as crystal lattice size decreases within nanoscale range
	Differential scanning calorimetry (DSC)	Change in heat flow due to sample changes during heat/cooling	Polymorphic form (melting temperature, crystallisation temperature) amorphous form (glass transition temperature), crystallinity (enthalpy of fusion, enthalpy of crystallisation, heat capacity change at glass transition temperature)	Thermogram, qualitative and quantitative	Modulated temperature DSC to separate overlapping irreversible and reversible thermal events, ultrafast heating	Powder form, few milligrams	Destructive. Results will be different with open or closed (hermetically sealed) pans	[18]
	Infrared (IR) spectroscopy (mid-IR spectroscopy)	Change in dipole moment during molecular vibrations	Polymorphic form (peak shifts and relative intensities), crystallinity (broadening of bands, peak shifts and relative intensities)	Spectrum, qualitative and quantitative, suitable for multivariate analysis	Diffuse reflectance IR (DRIFTS), attenuated total reflection (ATR), microscope	Powder or tablet form, depends on sampling setup, few milligrams. Wet samples usually problematic.	Sample preparation/measurement can involve pressure which can induce solid state transformations	[19,20]
	Raman spectroscopy	Change in polarisability during molecular vibrations	Polymorphic form, crystallinity	Spectrum, qualitative and quantitative, suitable for multivariate analysis	Various sample holders (within spectrometer, sampling probes, microscope)	Powder or suspension, few milligrams (usually). Fluorescent samples are problematic.	Sample heating can be problematic. Samples can be in aqueous medium.	[20,21,22]
Size and morphology	Dynamic light scattering (photon correlation spectroscopy)	Fluctuation of Rayleigh scattering of light associated with Brownian motion of nanoparticles	Particle size, particle size distribution	Particle size distribution (number based mean particle (hydrodynamic) size (Z-average), polydispersity index), quantitative		Suspension with suitable concentration	Suitable only for particles in nanometre size range	[23]
							Viscosity of suspension and temperature affect results
	Scanning electron microscopy (SEM)	Backscattering of electrons	Topographical information about particles	Scanning electron micrograph, particle morphology, size	Elemental analysis	Dry sample mounted on stage condition setup (vacuum), microgram requirement	Sample preparation destructive	[15]
	Transmission electron microscopy	Transmission of electrons	Density information	Transmission electron micrograph, morphology of cross sections, stabilizer- nanocrystal interaction		Embedded cross section preparation, microgram requirement	Sample preparation destructive	[24]
Surface properties	Zeta-potential	Dynamic electrophoretic mobility under electric field	Surface charge (zeta potential)	Zeta potential, quantitative		Suspension with suitable concentration		[25]
	Surface plasmon resonance (SPR)	Changes in refractive index in the vicinity of a planar sensor surface	Surface adsorption	Spectrum, interaction between stabiliser drug crystals, qualitative and quantitative		Substrate on planar surface sensor required (not direct measurement of nanocrystals)	Careful sample preparation required	[26]
Drug delivery	Dissolution testing	Dissolved drug analysed over time, usually using UV spectroscopy or HPLC	Dissolution profile	Solution concentration vs time	Paddle, flow through cell (with/without membrane insert), pharmacopeial/non pharmacopeial		Separating nanocrystals from dissolution medium can be problematic	[14]
	Fluorescence microscopy	Fluorescence by endogenous or added fluorophores	Localization of nanocrystals in relation to cells and tissues	Fluorescence (and nanocrystal) image	One or two photon (two photon fluorescence offers inherent confocality, sub-micron spatial resolution, deeper penetration in tissues) fluorescence	Non-fluorescent nanocrystals require fluorphore to physically entrapped into nanocrystals	Entrapment and leakage of fluorophore can be difficult or problematic	[17]
	Non-linear Raman microscopy	Change in polarisability during molecular vibrations.	Label free localisation of particles	Intensity of CARS shift (narrow band) or spectrum, (multiplex or broad band). Most commonly qualitative. 2D or 3D images.	Can be dry or aqueous suspension, in cell cultures or tissue samples	Coloured and two-photon fluorescent samples can interfere with signal. Can be coupled with other nonlinear phenomena such as second harmonic generation or two photon electronic fluorescence	Label free. Optimal lateral spatial resolution approximately 300–400 nm.	[21,27]