Environmental Engineering Reference
In-Depth Information
to organisms: when aggregates or heteroaggregates become too large for direct transport across the cell wall and/or membrane,
uptake may be prevented.
32.2.1.3 Experimental Techniques
Nanoparticulate systems are not uniquely composed by single discrete molecular
species, resulting in the appearance of specific artifacts that must mandatorily be taken into account. Consequently, the
identification and quantification of these systems need rigorous requirements relying on numerous analytical techniques and/or
methodologies in order to validate the obtained results [15]. Furthermore, appropriate traceable standards, quality control pro-
cedures, and, when available, standard reference materials must be used in order to validate the analytical techniques and/or
methodologies. Unfortunately, very few standard reference materials are available for NM analysis, and currently no standard
that consists of complex matrices with certified concentrations of NMs is available. This is in fact an area of great research but
remains a major challenge due to the inherent reactivity and instability of the NMs.
The quantification of the dissolution of NMs is not trivial. Besides the difficulties in adapting the existing analytical tech-
niques and/or methodologies, another important factor in the assessment of NM dissolution is the separation of particles from
the dissolved components. The flawed separation of the particles from the dissolved components results in over/underquantifi-
cation of the particles' dissolution. (Ultra)filtration [16], dialysis [17], or (ultra)centrifugation [18] methods are usually used to
separate the particles from the dissolved species. Dialysis and (ultra)filtration methods are membrane separation methods based
on physical separation that are dependent on the size of the membrane pores, and differ only in the driving force, which for
(ultra)filtration is the applied pressure and for dialysis the concentration gradient. A drawback is the size of the filters currently
available; the lowest are 20 nm pore size filters. The use of the combination of centrifugation and filtration (centrifugal ultrafil-
tration) has been recently seen as an effective way to overcome this problem due to the low pore sizes of the membranes avail-
able (cutoff value down to 3 kDa). Once the separation of particles from the dissolved components is ensured, a range of
analytical techniques can be applied to quantify the dissolution (inductively coupled plasma mass spectrometry (ICP-MS);
inductively coupled plasma atomic emission spectroscopy (ICP-AES), atomic absorption spectrometry (AAS), voltammetric
techniques). The choice of the analytical technique is based on the sensitivity and detection limit intended. The operation of the
ICP-MS instrument in the single-particle mode (SP-ICP-MS) [19] allows the quantification of the dissolved fraction, but with
the advantage of not requiring a separation process between particulates and dissolved species (discussed later). The optimal
recovery of the dissolved fraction is the main challenge for the effective separation of particles and dissolved species. This is a
still more critical challenge when stabilizers are used or when the adsorption of ligands/colloids in NMs occurs that have a
binding capacity through the dissolved species (discussed in the following section).
Many different state-of-the-art analytical techniques are available for size measurements including microscopy techniques,
dynamic light scattering (DlS), fluorescence correlation spectroscopy (FCS), nanoparticle tracking analysis (NTA), SP-ICP-MS,
and separation-based techniques such as field flow fractionation (FFF) or hydrodynamic chromatography (HDC). Each tech-
nique has its strengths and weaknesses and results can vary greatly according to the technique selected, the way it is used, and
the types of particles studied [15].
Microscopy techniques such as transmission electron microscopy (TEM) [20] and atomic force microscopy (AFM) [21] can
achieve sufficient spatial resolution, allowing the distinction of even the smallest single particles (1 nm resolution, which in AFM
is limited to the
z
dimension). For a large number of observations, TEM provides a number average diameter, and when coupled
with energy-dispersive spectrometry (EDSp) and electron diffraction, it can additionally provide information on the elemental
composition and crystal phase, respectively. For particles that are of similarly size or smaller than the radius of curvature of the
AFM tip, the measurement of the lateral distances is usually biased, and, therefore, the height measurements are considered to
be more accurate. The height quantifications will correspond to number average diameters when spherical particles are mea-
sured. Both microscopy techniques can provide reasonably accurate number average dimensions that have little experimental
bias when the sample preparation is free from artifacts, which can be a major challenge when nanoparticulate systems are studied
[15]. However, these techniques often suffer from tedious sample preparation, and low sample numbers coupled with high capital
and operating costs, especially when attempting to obtain quantitative data for a representative sample.
In DlS [22], one of the most used techniques for NM characterization, the light from a coherent source is scattered when it
is directed at the particle suspension. The scattering fluctuates with time due to the random Brownian motion of the particles,
allowing the determination of a
z
-averaged translational diffusion coefficient (
D
) from the autocorrelation of the Doppler shifts.
DlS has the advantage of being a nondestructive technique with a minimum of sample processing. An important drawback is
its strong dependence on the Rayleigh scattering on the particle radius (sixth power dependency). Consequently, the presence
of aggregates or even contaminating particles such as dust can mask the NPs' signal. The increase in particle scattering by
increasing their concentration, most often beyond those found in the environment [23], does not result in an advantage since the
consequence is often greater particle aggregation. However, DlS can be very useful in following the kinetics of NM aggregation
since it measures the gradual evolution of the
z
-average hydrodynamic diameter with time.
